Mitigation of epileptic seizures by combination therapy using benzodiazepines and neurosteroids

ABSTRACT

Provided are compositions comprising a benzodiazepine and a neurosteroid, containing one or both of the benzodiazepine and the neurosteroid in a subtherapeutic dose, and administration of such compositions for mitigation of an epileptic seizure. Further provided are compositions comprising a benzodiazepine, a neurosteroid, and an NMDA blocker, and administration of such compositions for mitigation of an epileptic seizure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/649,460 filed on Jul. 13, 2017, which is a continuation of U.S.application Ser. No. 13/964,922 filed on Aug. 12, 2013, now abandoned,which claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 61/682,745 filed on Aug. 13, 2012, and U.S. ProvisionalApplication No. 61/798,094 filed on Mar. 15, 2013, all of which arehereby incorporated herein by reference in their entireties for allpurposes.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under Grant Nos.AG032119, NS072094, and NS079202 awarded by the National institutes ofHealth. The Government has certain rights in this invention.

FIELD

Provided are compositions comprising a benzodiazepine and aneurosteroid, containing one or both of the benzodiazepine and theneurosteroid in a subtherapeutic dose, and administration of suchcompositions for mitigation of an epileptic seizure. Further providedare compositions comprising a benzodiazepine, a neurosteroid, and anNMDA blocker, and administration of such compositions for mitigation ofan epileptic seizure.

BACKGROUND

Tetramethylenedisulfotetramine (TETS), commonly called tetramine orTETS, is a highly toxic convulsant with a parenteral LD50 of 0.1-0.3mg/kg in mice or rats (Haskell and Voss, 1957; Voss et al., 1961; Casidaet al., 1976). In adult humans, 7-10 mg is estimated as a lethal dose(Guan et al., 1993) TETS was used as a rodenticide until bannedworldwide in the early 1990's (Whitlow et al., 2005. Banks et al.,2012). It is, however, still available illegally, and is responsible foraccidental and intentional poisonings, predominantly in China (Croddy,2004, Wu and Sun, 2004, Zhang et al. 2011), but also in other countries,including the United States (Barrueto et al., 2003). Between 1991 and2010 over 14,000 cases of TETS intoxication were reported in China with932 deaths (Li et al., 2011). Extreme toxicity, history of intentionalmass poisonings, and the absence of a specific antidote raise concernthat TETS is a potential chemical threat agent that could cause masscasualties if released accidentally or intentionally (Whitlow et al.,2005; Jell and Yeung, 2010).

Mild to moderate poisoning with TETS leads to headache and dizzinesswhereas severe intoxication produces status epilepticus and coma(Whitlow et al., 2005; Li et al., 2011). Animal studies demonstrate thatTETS is active as a convulsant when administered orally, parenterallyand intraventricularly. Sublethal seizures are not associated withevidence of cellular injury or neurodegeneration although there isdelayed transient reactive astrocytosis and microglial activation(Zolkowska et al., 2012).

The primary convulsant mechanism of TETS has been thought to relate toblockade of GABA_(A) receptors and the seizures induced in animalsresemble those produced by other GABA_(A) receptor antagonists includingpicrotoxin and pentylenetetrazol. Limited cellular physiological studiesand results from [³⁵S]t-butylbicyclophosphorothionate binding to brainmembranes indicate that TETS inhibits GABA_(A) receptors with an IC50 inthe range of 1 μM (Squires et al, 1983; Esser et al, 1991; Ratra et al.,2001) and it is therefore comparable in potency to picrotoxin as aninhibitor of GABA_(A) receptors (Squires et al., 1983; Cole and Casida,1986, Ratra et al, 2001).

Cultured hippocampal neurons display synchronous spontaneous Ca2+oscillations (Tanaka et al, 1996) that are driven by actionpotential-dependent synaptic transmission Disruption of Ca2+oscillations by environmental toxicants has been reported (Soria-Mercadoet al., 2009; Cao et al, 2010, Choi et al, 2010, Percira et al., 2010,Cao et al., 2011). Hippocampal neurons also exhibit spontaneouselectrical discharges as they form functional neuronal networks. Thesedischarges, as detected in extracellular recordings, consist ofinfrequent synchronized field potentials, mixed with more frequentdesynchronized random action potentials (Cao et al., 2012; Frega et al.,2012). Synchronous Ca2− oscillations and neuronal electrical firingco-occur (Jimbo et al., 1993) and are important in mediating neuronaldevelopment and activity dependent dendritic growth (Wayman et al.,2008) Genetic or environmental factors that interfere with neuronaltransmission influence the overall neuronal networks activity (Kenet etal., 2007, Meyer et al., 2008; Shafer et al., 2008; Frega et al., 2012;Wayman et al., 2012). For example picrotoxin, a GABA_(A) receptorantagonist, produces striking changes in network electric activity (Caoet al., 2012; Frega et al., 2012). Diisopropylfluorophosphate, anirreversible inhibitor of cholinesterase has also been shown to elicitstatus epileptics in rats. Hippocampal neurons dissociated from thebrains of diisopropylfluorophosphate exposed rats display significantlyhigher intracellular Ca2| concentration which appears to be dependent onthe N-methy-D-aspartate receptors (Deshpande et al., 2010).

In the present study, using rapid throughput assays we characterized theinfluence of TETS on the Ca2| dynamics and neuronal firing activity.Inasmuch as TETS induces changes in Ca2· dynamics that are similar tothose produced by the GABA_(A) receptor antagonists picrotoxin andbicuculline, our results support the view that TETS acts as a GABA_(A)receptor antagonist. Using rapid throughput Ca2· measurement, weidentified several agents that reduce or prevent the alterations in Ca2−dynamics induced by TETS, suggesting several treatment strategies forTETS-induced seizures, including the GABA_(A) receptor positivemodulators diazepam and allopregnanolone. In preliminary studies withmice, we confirmed that these two agents do inhibit TETS-induced clonicseizures and progression to tonic seizures and death supporting thatmeasurement of Ca2· dynamics is likely useful for identifying noveltargeted interventions for TETS poisoning.

SUMMARY

In one aspect, provided are compositions comprising a benzodiazepine anda neurosteroid. In varying embodiments, the compositions comprise one orboth of the benzodiazepine and the neurosteroid in a subtherapeutic doseor amount. In varying embodiments, the compositions further comprise aNMDA receptor antagonist. In some embodiments, the composition isformulated for inhalational, intranasal or intrapulmonaryadministration. In some embodiments, the composition is formulated fororal or transmucosal delivery. In varying embodiments, the compositionis formulated for parenteral delivery. In some embodiments, theparenteral delivery or administration is via a route selected from thegroup consisting of inhalational, intrapulmonary, intranasal,intramuscular, subcutaneous, transmucosal and intravenous. In someembodiments, the composition is formulated for intramuscular delivery.In some embodiments, the benzodiazepine is an agonist of thebenzodiazepine recognition site on GABA_(A) receptors and stimulatesendogenous neurosteroid synthesis. In some embodiments, thebenzodiazepine is selected from the group consisting of bretazenil,clonazepam, cloxazolam, clorazepate, diazepam, fludiazepam,flutoprazepam, lorazepam, midazolam, nimetazepam, nitrazepam,phenazepam, temazepam and clobazam. In some embodiments, thebenzodiazepine is selected from the group consisting of midazolam,lorazepam and diazepam. In some embodiments, the neurosteroid isselected from the group consisting of allopregnanolone,allotetrahydrodeoxycorticosterone, ganaxolone, alphaxolone, alphadolone,hydroxydione, minaxolone, and Althesin. In some embodiments, theneurosteroid is allopregnanolone. In some embodiments, the compositioncomprises allopregnanolone and a benzodiazepine selected from the groupconsisting of midazolam, lorazepam and diazepam. In some embodiments,the neurosteroid is suspended or dissolved in a cyclodextrin (e.g., anα-cyclodextrin, a β-cyclodextrin or a γ-cyclodextrin). In varyingembodiments, the neurosteroid is suspended or dissolved in acyclodextrin selected from the group consisting ofhydroxypropyl-β-cyclodextrin, endotoxin controlled β-cyclodextrinsulfobutyl ethers, or cyclodextrin sodium salts (e.g., CAPTISOL®). Insome embodiments, the neurosteroid is suspended or dissolved in anedible oil. In some embodiments, the edible oil comprises one or morevegetable oils. In some embodiments, the vegetable oil is selected fromthe group consisting of coconut oil, corn oil, cottonseed oil, oliveoil, palm oil, peanut oil, rapeseed oil, canola oil, safflower oil,sesame oil, soybean oil, sunflower oil, and mixtures thereof. In someembodiments, the edible oil is canola oil. In some embodiments, theedible oil comprises one or more nut oils. In some embodiments, the nutoil is selected from the group consisting of almond oil, cashew oil,hazelnut oil, macadamia oil, mongongo nut oil, pecan oil, pine nut oil,pistachio oil, walnut oil, and mixtures thereof. In some embodiments,the NMDA receptor antagonist is selected from the group consisting ofdizocilpine (MK-801), meperidine, methadone, dextropropoxyphene,tramadol, ketobemidone, ketamine, dextromethorphan, phencyclidine,nitrous oxide (N₂O), AP5 (R-2-amino-5-phosphonopentanoate), AP7(2-amino-7-phosphonoheptanoic acid), CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,eticyclidine, gacyclidine, ibogaine, magnesium, memantine,methoxetamine, rolicyclidine tenocyclidine, methoxydine, tiletamine,xenon, neramexane, cliprodil, etoxadrol, dexoxadrol, WMS 2539, NEFA,remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211, rhynchophylline,1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA(5,7-dichlorokynurenic acid), kynurenic acid, lacosamide, CP-101,606(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some embodiments,the NMDA receptor antagonist is selected from the group consisting ofketamine, dextromethorphan, phencyclidine. CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine dextrorphan, memantine, tiletamine, neramexane,cliprodil, remacemide, aptiganel, 1-Aminocyclopropanecarboxylic acid(ACPC), 7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenicacid, CP-101,606 (traxoprodil), AZD6765 (lanicemine) and GLYX-13. Insome embodiments, the NMDA receptor antagonist is dizocilpine (MK-801).In varying embodiments, the composition comprises a benzodiazepine and aneurosteroid formulated in a cyclodextrin, e.g., for intramuscular,intravenous and/or subcutaneous administration.

In another aspect, provided are methods of preventing or terminating aseizure in a subject in need thereof. In varying embodiments, themethods comprise administration to the subject of an effective amount ofa composition as described above and herein. Also provided are methodsof accelerating the termination or abortion of an impending seizure in asubject in need thereof. In varying embodiments, the methods compriseadministration to the subject of an effective amount of a composition asdescribed above and herein. In some embodiments, the composition isadministered via inhalational or intrapulmonary administration. In someembodiments, the composition is not heated prior to administration. Insome embodiments, the composition is nebulized. In some embodiments, thenebulized particles are about 3 μm or smaller. In some embodiments, thenebulized particles are about 2-3 μm. In some embodiments, thecomposition is delivered to the distal alveoli. In some embodiments, thecomposition is administered orally. In some embodiments, the compositionis contained within a soft gel capsule. In some embodiments, thecomposition is administered parenterally. In some embodiments, thecomposition is administered via a parenteral route selected from thegroup consisting of inhalational, intrapulmonary, intranasal,intramuscular, subcutaneous, transmucosal and intravenous. In someembodiments, the composition is administered transmucosally. In varyingembodiments, the method comprises co-administering a benzodiazepine anda neurosteroid formulated in a cyclodextrin, e.g., intramuscularly,intravenously and/or subcutaneously.

In a related aspect, methods of preventing or terminating a seizure in asubject in need thereof, comprising administration to the subject of aneffective amount of a benzodiazepine and a neurosteroid. In varyingembodiments, one or both of the benzodiazepine and the neurosteroid areadministered in a subtherapeutic dose. Further are provided methods ofaccelerating the termination or abortion of an impending seizure in asubject in need thereof. In varying embodiments, the methods compriseadministration to the subject of an effective amount of a benzodiazepineand a neurosteroid. In some embodiments, one or both of thebenzodiazepine and the neurosteroid are administered in a subtherapeuticdose. In some embodiments, the benzodiazepine and the neurosteroid areco-administered together and/or by the same route of administration. Insome embodiments, the benzodiazepine and the neurosteroid areco-administered separately and/or by different routes of administration.In some embodiments, one or both of the benzodiazepine and theneurosteroid are self-administered by the subject. In some embodiments,one or both of the benzodiazepine and the neurosteroid are administeredvia inhalational or intrapulmonary administration. In some embodiments,one or both of the benzodiazepine and the neurosteroid are not heatedprior to administration. In some embodiments, one or both of thebenzodiazepine and the neurosteroid are nebulized. In some embodiments,the nebulized particles are about 3 μm or smaller. In some embodiments,the nebulized particles are about 2-3 μm. In some embodiments, one orboth of the benzodiazepine and the neurosteroid are delivered to thedistal alveoli. In some embodiments, one or both of the benzodiazepineand the neurosteroid are administered orally. In some embodiments, oneor both of the benzodiazepine and the neurosteroid are contained withina soft gel capsule. In some embodiments, one or both of thebenzodiazepine and the neurosteroid are administered parenterally. Insome embodiments, one or both of the benzodiazepine and the neurosteroidare administered via a parenteral route selected from the groupconsisting of inhalational, intrapulmonary, intranasal, intramuscular,subcutaneous, transmucosal and intravenous. In some embodiments, one orboth of the benzodiazepine and the neurosteroid are administeredtransmucosally. In varying embodiments, the method comprisesco-administering a benzodiazepine and a neurosteroid formulated in acyclodextrin, e.g., intramuscularly, intravenously and/orsubcutaneously.

With respect to further embodiments of the methods, in some embodiments,the benzodiazepine is selected from the group consisting of bretazenil,clonazepam, cloxazolam, clorazepate, diazepam, fludiazepam,flutoprazepam, lorazepam, midazolam, nimetazepam, nitrazepam,phenazepam, temazepam and clobazam. In some embodiments, thebenzodiazepine is selected from the group consisting of midazolam,lorazepam, and diazepam. In some embodiments, the benzodiazepine isadministered at a dose in the range of 0.3 μg/kg to 3.0 μg/kg. Invarying embodiments, the benzodiazepine is administered at a dose thatdoes not decrease blood pressure. In some embodiments, the neurosteroidis selected from the group consisting of allopregnanolone,allotetrahydrodeoxycorticosterone, ganaxolone, alphaxolone, alphadolone,hydroxydione, minaxolone, and Althesin. In some embodiments,allopregnanolone is co-administered with a benzodiazepine selected fromthe group consisting of midazolam, lorazepam, and diazepam. In someembodiments, the methods further comprise co-administration of an NMDAreceptor antagonist. In some embodiments, the NMDA receptor antagonistis selected from the group consisting of dizocilpine (MK-801),meperidine, methadone, dextropropoxyphene, tramadol, ketobemidone,ketamine, dextromethorphan, phencyclidine, nitrous oxide (N₂O), AP5(R-2-amino-5-phosphonopentanoate), AP7 (2-amino-7-phosphonoheptanoicacid), CPPene (3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonicacid), selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,eticyclidine, gacyclidine, ibogaine, magnesium, memantine,methoxetamine, rolicyclidine tenocyclidine, methoxydine, tiletamine,xenon, neramexane, eliprodil, etoxadrol, dexoxadrol, WMS 2539, NEFA,remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211, rhynchophylline,1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA(5,7-dichlorokynurenic acid), kynurenic acid, lacosamide, CP-101,606(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some embodiments,the NMDA receptor antagonist is selected from the group consisting ofketamine, dextromethorphan, phencyclidine, CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine, dextrorphan, memantine, tiletamine, neramexane,cliprodil, ramacemide, aptiganel, 1-Aminocyclopropanecarboxylic acid(ACPC). 7-Chlorokynurenate. DCKA (5,7-dichlorokynurenic acid), kynurenicacid, CP-101,606 (traxoprodil), AZD6765 (lanicemine) and GLYX-13. Insome embodiments, the subject is experiencing aura. In some embodiments,the subject has been warned of an impending seizure. In someembodiments, the subject is experiencing a seizure. In some embodiments,the subject has status epilepticus, refractory status epilepticus orsuper-refractory status epilepticus. In some embodiments, the subjecthas myoclonic epilepsy. In some embodiments, the subject suffers fromseizure clusters. In some embodiments, the seizure is a tonic seizure.In some embodiments, the seizure is a clonic seizure. In someembodiments, the subject has been exposed to or is at risk of beingexposed to a nerve agent or a pesticide that can cause seizures. In someembodiments, the subject has been exposed to or is at risk of beingexposed to tetramethylenedisulfotetramine (TETS).

In another aspect, further provided are kits comprising a benzodiazepineand a neurosteroid. In varying embodiments, one or both of thebenzodiazepine and the neurosteroid are provided in unit dosage formscomprising a subtherapeutic dose. In some embodiments, the kits furthercomprise a NMDA receptor antagonist. In some embodiments, one or both ofthe benzodiazepine and the neurosteroid is formulated for inhalational,intranasal or intrapulmonary administration. In some embodiments, one orboth of the benzodiazepine and the neurosteroid is formulated for oralor parenteral delivery. In some embodiments, one or both of thebenzodiazepine and the neurosteroid are formulated for a parenteralroute selected from the group consisting of inhalational,intrapulmonary, intranasal, intramuscular, subcutaneous, transmucosaland intravenous. In some embodiments, the benzodiazepine is an agonistof the benzodiazepine recognition site on GABA_(A) receptors andstimulates endogenous neurosteroid synthesis. In some embodiments, thebenzodiazepine is selected from the group consisting of bretazenil,clonazepam, cloxazolam, clorazepate, diazepam, fludiazepam,flutoprazepam, lorazepam, midazolam, nimetazepam, nitrazepam,phenazepam, temazepam and clobazam. In some embodiments, thebenzodiazepine is selected from the group consisting of midazolam,lorazepam and diazepam. In some embodiments, the neurosteroid isselected from the group consisting of allopregnanolone,allotetrahydrodeoxycorticosterone, ganaxolone, alphaxolone, alphadolone,hydroxydione, minaxolone, and Althesin. In some embodiments, theneurosteroid is allopregnanolone. In some embodiments, the kit comprisesallopregnanolone and a benzodiazepine selected from the group consistingof midazolam, lorazepam, and diazepam. In some embodiments, theneurosteroid is suspended or dissolved in a cyclodextrin (e.g., anα-cyclodextrin, a β-cyclodextrin or a γ-cyclodextrin). In varyingembodiments, the neurosteroid is suspended or dissolved in acyclodextrin selected from the group consisting ofhydroxypropyl-β-cyclodextrin, endotoxin controlled β-cyclodextrinsulfobutyl ethers, or cyclodextrin sodium salts (e.g., CAPTISOL®). Insome embodiments, the neurosteroid is suspended or dissolved in anedible oil. In some embodiments, the edible oil comprises one or morevegetable oils. In some embodiments, the vegetable oil is selected fromthe group consisting of coconut oil, corn oil, cottonseed oil, oliveoil, palm oil, peanut oil, rapeseed oil, canola oil, safflower oil,sesame oil, soybean oil, sunflower oil, and mixtures thereof. In someembodiments, the edible oil is canola oil. In some embodiments, theedible oil comprises one or more nut oils. In some embodiments, the nutoil is selected from the group consisting of almond oil, cashew oil,hazelnut oil, macadamia oil, mongongo nut oil, pecan oil, pine nut oil,pistachio oil, walnut oil, and mixtures thereof. In some embodiments,the NMDA receptor antagonist is selected from the group consisting ofdizocilpine (MK-801), meperidine, methadone, dextropropoxyphene,tramadol, ketobemidone, ketamine, dextromethorphan, phencyclidine,nitrous oxide (N₂O), AP5 (R-2-amino-5-phosphonopentanoate), AP7(2-amino-7-phosphonoheptanoic acid), CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,eticyclidine, gacyclidine, ibogaine, magnesium, memantine,methoxetamine, rolicyclidine, tenocyclidine, methoxydine, tiletamine,xenon, neramexane, eliprodil, etoxadrol, dexoxadrol, WMS 2539, NEFA,remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211, rhynchophylline,1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA(5,7-dichlorokynurenic acid), kynurenic acid, lacosamide, CP-101,606(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some embodiments,the NMDA receptor antagonist is selected from the group consisting ofketamine, dextromethorphan, phencyclidine. CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine, dextrorphan, memantine, tiletamine, neramexane,eliprodil, remacemide, aptiganel, 1-Aminocyclopropanecarboxylic acid(ACPC), 7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenicacid, CP-101,606 (traxoprodil), AZD6765 (lanicemine) and GLYX-13. Insome embodiments, the NMDA receptor antagonist is dizocilpine (MK-801).

In another aspect, the invention provides compositions comprising abenzodiazepine, a neurosteroid and an NMDA receptor antagonist. In someembodiments, the composition is formulated for inhalational orintrapulmonary administration. In some embodiments, the composition isformulated for oral or transmucosal delivery. In some embodiments, thebenzodiazepine is an agonist of the benzodiazepine recognition site onGABA_(A) receptors and stimulates endogenous neurosteroid synthesis. Insome embodiments, the benzodiazepine is selected from the groupconsisting of bretazenil, clonazepam, cloxazolam, clorazepate, diazepam,fludiazepam, flutoprazepam, lorazepam, midazolam, nimetazepam,nitrazepam, phenazepam, temazepam and clobazam. In some embodiments, thebenzodiazepine is midazolam. In some embodiments, the benzodiazepine isdiazepam. In some embodiments, the neurosteroid is selected from thegroup consisting of allopregnanolone, allotetrahydrodeoxycorticosterone,ganaxolone, alphaxolone, alphadolone, hydroxydione, minaxolone, andAlthesin. In some embodiments, the neurosteroid is allopregnanolone. Insome embodiments, the neurosteroid is suspended or dissolved in acyclodextrin (e.g., an α-cyclodextrin, a β-cyclodextrin or aγ-cyclodextrin). In varying embodiments, the neurosteroid is suspendedor dissolved in a cyclodextrin selected from the group consisting ofhydroxypropyl-β-cyclodextrin, endotoxin controlled β-cyclodextrinsulfobutyl ethers, or cyclodextrin sodium salts (e.g., CAPTISOL®). Insome embodiments, the neurosteroid is suspended or dissolved in anedible oil. In some embodiments, the edible oil comprises one or more avegetable oils. In some embodiments, the vegetable oil is selected fromthe group consisting of coconut oil, corn oil, cottonseed oil, oliveoil, palm oil, peanut oil, rapeseed oil, canola oil, safflower oil,sesame oil, soybean oil, sunflower oil, and mixtures thereof. In someembodiments, the edible oil is canola oil. In some embodiments, theedible oil comprises one or more nut oils. In some embodiments, the nutoil is selected from the group consisting of almond oil, cashew oil,hazelnut oil, macadamia oil, mongongo nut oil, pecan oil, pine nut oil,pistachio oil, walnut oil, and mixtures thereof in some embodiments, theNMDA receptor antagonist is selected from the group consisting ofdizocilpine (MK-801), meperidine, methadone, dextropropoxyphene,tramadol, ketobemidone, ketamine, dextromethorphan, phencyclidine,nitrous oxide (N₂O), AP5 (R-2-amino-5-phosphonopentanoate), AP7(2-amino-7-phosphonoheptanoic acid), CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,eticyclidine, gacyclidine, ibogaine, magnesium, memantine,methoxetamine, rolicyclidine tenocyclidine, methoxydine, tiletamine,xenon, neramexane, cliprodil, etoxadrol, dexoxadrol, WMS 2539, NEFA,remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211, rhynchophylline,1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA(5,7-dichlorokynurenic acid), kynurenic acid, lacosamide, CP-101,606(traxoprodil). AZD6765 (lanicemine) and GLYX-13. In some embodiments,the NMDA receptor antagonist is selected from the group consisting ofketamine, dextromethorphan, phencyclidine, CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine, dextrorphan, memantine, tiletamine, neramexane,eliprodil, remacemide, aptiganel, 1-Aminocyclopropanecarboxylic acid(ACPC), 7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenicacid, CP-101,606 (traxoprodil), AZD6765 (lanicemine) and GLYX-13.

In another aspect, the invention provides methods of preventing orterminating a seizure in a subject in need thereof, comprisingadministration to the subject of an effective amount of a composition asdescribed above and herein. In another aspect, the invention providesmethods of accelerating the termination or abortion of an impendingseizure in a subject in need thereof, comprising administration to thesubject of an effective amount of a composition as described above andherein. In a further aspect, the invention provides methods ofpreventing or terminating a seizure in a subject in need thereof,comprising administration to the subject of an effective amount of abenzodiazepine, a neurosteroid and an NMDA receptor antagonist. In afurther aspect, the invention provides methods of accelerating thetermination or abortion of an impending seizure in a subject in needthereof, comprising administration to the subject of an effective amountof a benzodiazepine, a neurosteroid and an NMDA receptor antagonist. Insome embodiments, the benzodiazepine, neurosteroid and NMDA receptorantagonist are co-administered together and/or by the same route ofadministration. In some embodiments, the benzodiazepine, neurosteroidand NMDA receptor antagonist are co-administered separately and/or bydifferent routes of administration. In some embodiments, thebenzodiazepine is selected from the group consisting of bretazenil,clonazepam, cloxazolam, clorazepate, diazepam, fludiazepam,flutoprazepam, lorazepam, midazolam, nimetazepam, nitrazepam,phenazepam, temazepam and clobazam. In some embodiments, theneurosteroid is selected from the group consisting of allopregnanolone,allotetrahydrodeoxycorticosterone, ganaxolone, alphaxolone, alphadolone,hydroxydione, minaxolone, and Althesin. In some embodiments, the NMDAreceptor antagonist is selected from the group consisting of dizocilpine(MK-801), meperidine, methadone, dextropropoxyphene, tramadol,ketobemidone, ketamine, dextromethorphan, phencyclidine, nitrous oxide(N₂O), AP5 (R-2-amino-5-phosphonopentanoate), AP7(2-amino-7-phosphonoheptanoic acid). CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,eticyclidine, gacyclidine, ibogaine, magnesium, memantine,methoxetamine, rolicyclidine.tenocyclidine, methoxydine, tiletamine,xenon, neramexane, eliprodil, etoxadrol, dexoxadrol, WMS 2539, NEFA,remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211, rhynchophylline,1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA(5,7-dichlorokynurenic acid), kynurenic acid, lacosamide, CP-101,606(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some embodiments,the NMDA receptor antagonist is selected from the group consisting ofketamine, dextromethorphan, phencyclidine, CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine, dextrorphan, memantine, tiletamine, neramexane,eliprodil, remacemide, aptiganel, 1-Aminocyclopropanecarboxylic acid(ACPC), 7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenicacid, CP-101,606 (traxoprodil), AZD6765 (lanicemine) and GLYX-13. In oneembodiment, the subject is experiencing aura. In one embodiment, thesubject has been warned of an impending seizure. In one embodiment, thesubject is experiencing a seizure. In one embodiment, the subject hasstatus epilepticus, refractory status epilepticus or super-refractorystatus epilepticus. In one embodiment, the subject has myoclonicepilepsy. In one embodiment, the subject suffers from seizure clusters.In one embodiment, the seizure is a tonic seizure. In one embodiment,the seizure is a clonic seizure. In one embodiment, the benzodiazepineis self-administered by the subject. In one embodiment, the compositionis administered via inhalational or intrapulmonary administration. Inone embodiment, the composition is not heated prior to administration.In one embodiment, the benzodiazepine is nebulized. In one embodiment,the nebulized particles are about 3 μm or smaller. In one embodiment,the nebulized particles are about 2.3 μm. In one embodiment, thebenzodiazepine is delivered to the distal alveoli. In one embodiment,the benzodiazepine is administered at a dose in the range of 0.3 μg/kgto 3.0 μg/kg. In varying embodiments, the benzodiazepine is administeredat a dose that does not decrease blood pressure. In one embodiment, thecomposition is administered orally. In one embodiment, the compositionis contained within a soft gel capsule. In one embodiment, thecomposition is administered transmucosally. In various embodiments, thesubject may be at risk of exposure to or may have been exposed totetramethylenedisulfotetramine (TETS).

Definitions

As used herein, “administering” refers to local and systemicadministration, e.g., including enteral, parenteral, pulmonary, andtopical/transdermal administration. Routes of administration for theagents (e.g., one or more of a benzodiazepine, a neurosteroid and/or anNMDA receptor antagonist) that find use in the methods described hereininclude, e.g., oral (per os (P.O.)) administration, nasal or inhalationadministration, administration as a suppository, topical contact,transdermal delivery (e.g., via a transdermal patch), intrathecal (IT)administration, intravenous (“iv”) administration, intraperitoneal(“ip”) administration, intramuscular (“im”) administration,intralesional administration, or subcutaneous (“sc”) administration, orthe implantation of a slow-release device e.g., a mini-osmotic pump, adepot formulation, etc., to a subject. Administration can be by anyroute including parenteral and transmucosal (e.g., oral, nasal, vaginal,rectal, or transdermal). Parenteral administration includes, e.g.,intravenous, intramuscular, intra-arterial, intradermal, subcutaneous,intraperitoneal, intraventricular, iontophoretic and intracranial. Othermodes of delivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The terms “systemic administration” and “systemically administered”refer to a method of administering a compound or composition to a mammalso that the compound or composition is delivered to sites in the body,including the targeted site of pharmaceutical action, via thecirculatory system. Systemic administration includes, but is not limitedto, oral, intranasal, rectal and parenteral (e.g., other than throughthe alimentary tract, such as intramuscular, intravenous,intra-arterial, transdermal and subcutaneous) administration.

The term “co-administration” refers to the presence of both activeagents in the blood at the same time. Active agents that areco-administered can be delivered concurrently (i.e., at the same time)or sequentially.

The phrase “cause to be administered” refers to the actions taken by amedical professional (e.g., a physician), or a person controllingmedical care of a subject, that control and/or permit the administrationof the agent(s)/compound(s) at issue to the subject. Causing to beadministered can involve diagnosis and/or determination of anappropriate therapeutic or prophylactic regimen, and/or prescribingparticular agent(s)/compounds for a subject. Such prescribing caninclude, for example, drafting a prescription form, annotating a medicalrecord, and the like.

The term “effective amount” or “pharmaceutically effective amount” referto the amount and/or dosage, and/or dosage regime of one or morecompounds necessary to bring about the desired result e.g., an amountsufficient prevent, abort or terminate a seizure.

“Sub-therapeutic dose” refers to a dose of a pharmacologically activeagent(s), either as an administered dose of pharmacologically activeagent, or actual level of pharmacologically active agent in a subjectthat functionally is insufficient to elicit the intended pharmacologicaleffect in itself (e.g., to abort or prevent a seizure), or thatquantitatively is less than the established therapeutic dose for thatparticular pharmacological agent (e g, as published in a referenceconsulted by a person of skill, for example, doses for a pharmacologicalagent published in the Physicians' Desk Reference, 67th Ed, 2013,Thomson Healthcare or Brunton, et al., Goodman & Gilman's ThePharmacological Basis of Therapeutics, 12th edition, 2010, McGraw-HillProfessional). A “sub-therapeutic dose” can be defined in relative terms(i.e., as a percentage amount (less than 100%) of the amount ofpharmacologically active agent conventionally administered). Forexample, a sub-therapeutic dose amount can be about 1% to about 75% ofthe amount of pharmacologically active agent conventionallyadministered. In some embodiments, a sub-therapeutic dose can be lessthan about 75%, 50%, 30%, 25%, 20%, 10% or less, than the amount ofpharmacologically active agent conventionally administered Asub-therapeutic dose amount can be in the range of about 1% to about 75%of the amount of pharmacologically active agent known to elicit theintended pharmacological effect. In some embodiments, a sub-therapeuticdose can be less than about 75%, 50%, 30%, 25%, 20%, 10% or less, thanthe amount of pharmacologically active agent known to elicit theintended pharmacological effect.

As used herein, the terms “treating” and “treatment” refer to delayingthe onset of, retarding or reversing the progress of, reducing theseverity of, or alleviating or preventing either the disease orcondition to which the term applies, or one or more symptoms of suchdisease or condition.

The term “mitigating” refers to reduction or elimination of one or moresymptoms of that pathology or disease, and/or a reduction in the rate ordelay of onset or severity of one or more symptoms of that pathology ordisease, and/or the prevention of that pathology or disease.

The terms “reduce,” “inhibit,” “relieve,” “alleviate” refer to thedetectable decrease in the frequency, severity and/or duration ofseizures. A reduction in the frequency, severity and/or duration ofseizures can be measured by self-assessment (e.g., by reporting of thepatient) or by a trained clinical observer. Determination of a reductionof the frequency, severity and/or duration of seizures can be made bycomparing patient status before and after treatment.

As used herein, the phrase “consisting essentially of” refers to thegenera or species of active pharmaceutical agents (e.g., neurosteroid incombination with benzodiazepine, optionally in further combination withan NMDA blocker) and excipient (e.g., a cyclodextrin, an edible oil)included in a method or composition. In various embodiments, otherunmentioned or unrecited active ingredients and inactive are expresslyexcluded. In various embodiments, additives (e.g., surfactants, acids(organic or fatty), alcohols, esters, co-solvents, solubilizers, lipids,polymers, glycols) are expressly excluded.

The terms “subject,” “individual,” and “patient” interchangeably referto a mammal, preferably a human or a non-human primate, but alsodomesticated mammals (e.g., canine or feline), laboratory mammals (e.g.,mouse, rat, rabbit, hamster, guinea pig) and agricultural mammals (e.g.,equine, bovine, porcine, ovine). In various embodiments, the subject canbe a human (e.g., adult male, adult female, adolescent male, adolescentfemale, male child, female child) under the care of a physician or otherhealthworker in a hospital, psychiatric care facility, as an outpatient,or other clinical context. In certain embodiments the subject may not beunder the care or prescription of a physician or other healthworker.

The term “edible oil” refers to an oil that is digestible by a mammal.Preferred oils are edible or digestible without inducing undesirableside effects.

The term “neuroactive steroid” or “neurosteroid” refers to steroidcompounds that rapidly alter neuronal excitability through interactionwith neurotransmitter-gated ion channels. Neurosteroids act asallosteric modulators of neurotransmitter receptors, such as GABA_(A),NMDA, and sigma receptors. Neurosteroids find use as sedatives for thepurpose of general anaesthesia for carrying out surgical procedures, andin the treatment of epilepsy and traumatic brain injury. Illustrativeneurosteroids include, e.g., allopregnanolone, Ganaxolone, alphaxolone,alphadolone, hydroxydione, minaxolone, and Althesin (a mixture ofalphaxolone and alphadolone).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D illustrate TETS-induced Ca2− dysregulation in hippocampalneurons. (A) Representative traces showing how acute exposure to TETS(0.1-10 μM) influences Ca2· fluctuations in hippocampal neurons 13-17DIV. Note that neurons exhibit spontaneous synchronous Ca2| oscillationsat this developmental stage indicative of functional networkconnectivity. The effects of TETS were analyzed in the initial 5 minfollowing addition (Phase I) and in the subsequent 10 min (Phase II). InPhase I, the integrated intracellular Ca2+ level increased in aconcentration-dependent fashion (B), and there was a plateau response athigher concentrations (3, 10 μM) that decayed slowly over the 5 minperiod. In Phase II, there was a concentration-dependent reduction inthe frequency and an increase in the amplitude of the spontaneoussynchronized Ca2− oscillations (C,D). The traces shown for Phase II arerepresentative samples of the 10 min Phase II period. This experimentwas repeated three times with similar results.

FIG. 2 illustrates reversal of TETS-induced Phase II effects afterwashout of TETS Traces show synchronized Ca2− oscillations that arereduced in frequency and increased in amplitude in the presence of TETS.The dotted red line is a representative trace before (“Phase IIresponse”) and after TETS (“Washout”). The solid black line is arepresentative recording from a control experiment in which the culturewas treated with vehicle and subjected to the same washout procedure.

FIGS. 3A-D illustrate TETS, picrotoxin, and bicuculline trigger similarneuronal Ca2+ dysregulation. (A) Representative traces from experimentscomparing the effects of TETS (3 μM), picrotoxin (100 μM), andbicuculline (100 μM) on Ca2+ fluctuations. The three agents producesimilar acute elevation of the integrated Ca2+ level (B) with plateauresponses in Phase I, and they decreased the oscillator) frequency (C)while increasing the amplitude of Ca2+ transients in Phase II (D). **,p<0.01, inhibitors vs. control, data were pooled from three experimentsperformed at least in duplicate.

FIGS. 4A-B illustrate TETS-reversibly alters spontaneous electricaldischarges in hippocampal neurons. (A) Representative raster plots ofneuronal discharges before, during and after exposure to vehicle (DMSO)(left panels) or TETS (right panels). Neuronal network activity wasstable for up to 60 min in the absence or presence of vehicle control.TETS solutions of increasing concentration were added serially to thewells. After recording for 10 min, the solution was removed and replacedby a higher concentration or by vehicle (wash out). TETS concentrationsof 2 and 6 μM caused a clustered burst discharge pattern and increasedthe overall discharge frequency (B). This experiment was repeated threetimes each performed in duplicate with similar results. *, p<0.05, **,p<0.01, TETS vs. basal.

FIGS. 5A-B illustrates TETS-induced a pattern of clustered electricalburst firing in hippocampal neuronal cell cultures at 14 days in vitro.Representative traces of neuronal electrical firing from an MEArecording before (A) and after (B) addition of TETS (6 μM). The softwareonly allows a display of 200 ms; the actual total period of clusteredbursts after TETS treatment often lasted up to 10 s (see FIG. 3A, rightpanel, 4th row).

FIGS. 6A-D illustrate MK-801, but not nifedipine, partially mitigatesTETS (3 μM)-induced neuronal Ca2+ dysregulation. (A) Representativetraces illustrating effects of pre-exposure to MK-801 and nifedipine onTETS-induced Ca2+ dysregulation. (B) Effects of MK-801 (MK) andnifedipine (NIF) on TETS-induced increase in integrated Ca2+ levels inPhase I. (C,D) Effects of MK-801 and nifedipine on the TETS-inducedsynchronous Ca2+ transient oscillation frequency decrease (C) andamplitude increase (D) in Phase II. **, p<0.01, TETS vs. vehiclecontrol, ##, p<0.01, MK-801+TETS vs TETS, n=6 pooled from twoexperiments.

FIGS. 7 A-D Diazepam partially mitigates TETS-induced neuronal Ca2+dysregulation. (A) Representative traces illustrating effects ofpre-exposure to increasing concentrations of diazepam (0.03-1 μM) onTETS-induced Ca2+ dysregulation. (B) Effect of diazepam (DZP) onTETS-induced increase in integrated Ca2+ levels in Phase I. (C,D) Effectof diazepam on the TETS-induced synchronous Ca2+ transient oscillationfrequency decrease (C) and amplitude increase (D) in Phase II. **,p<0.01, TETS vs. vehicle control, #, p<0.05, ##, p<0.01, diazepam+TETSvs. TETS, n=6 pooled from two experiments.

FIGS. 8A-D illustrate allopregnanolone partially mitigates TETS-inducedneuronal Ca2+ signaling dysregulation. (A) Representative tracesillustrating effects of pre-exposure to increasing concentrations ofallopregnanolone (0.03-1 μM) on TETS-induced Ca2+ dysregulation. (B)Effect of allopregnanolone (AlloP) on TETS-induced increase inintegrated Ca2+ levels in Phase I. (C,D) Effect of allopregnanolone onthe TETS-induced synchronous Ca2+ transient oscillation frequencydecrease (C) and amplitude increase (D) in Phase II. **, p<0.01, TETSvs. vehicle control, #, p<0.05, ##, p<0.01, allopregnanolone+TETS vsTETS, n=6 pooled from two experiments.

FIGS. 9A-D illustrate low concentrations of allopregnanolone anddiazepam in combination act synergistically to mitigate TETS-inducedneuronal Ca2+ signaling dysregulation. (A) Representative tracesillustrating effect of pre-exposure to allopregnanolone (0.1 μM),diazepam (0.1 μM) or a combination of allopregnanolone (0.1 μM) anddiazepam (0.1 μM) on TETS-induced Ca2+ dysregulation. (C,D) Effects ofallopregnanolone or diazepam alone or the combination on TETS-inducedsynchronous Ca2+ transient oscillation frequency decrease (C) andamplitude increase (D) in phase II. **, p<0.01, TETS vs. vehiclecontrol, ##, p<0.01, allopregnanolone/diazepam+TETS vs TETS, n=8 pooledfrom two experiments.

FIGS. 10A-C illustrate that exposure of mouse hippocampal neuronsfollowing TETS challenge with diazepam (0.1 μM) and allopregnanolone(0.1 μM) in combination effectively mitigates TETS dysregulated Ca2+dynamics. (A) Representative traces illustrating effects of post-TETStreatment with diazepam or allopregnanolone or the combination onTETS-induced Ca²⁺ dysregulation. Amelioration of TETS-inducedalterations in the Phase II response (see, Cao et al, ToxicologicalSciences (2012) 130:362-372) by diazepam and allopregnanolone, eithersingly or in combination, on the frequency of synchronous Ca²⁺oscillation (B) and increases in Ca²⁺ transient amplitude (C). The firstarrowhead indicates the addition of TETS or vehicle. The secondarrowhead indicates the addition of vehicle or diazepam orallopregnanolone or the combination. Each data point representsMean±SEM, n=6 wells.

FIG. 11 illustrates that high dose diazepam rescues animals fromTETS-induced tonic seizures and death. Representative EEG recordingsfrom mice administered TETS (0.15 mg/kg, i.p.) with and withoutpretreatment with diazepam (dose/route). Time to seizure onset andseizure duration are expressed as the mean±S.E.M. (n=x per treatmentgroup). Administration of diazepam immediately following the secondclonic seizure prevented a fatal tonic seizure. EEG recording inTETS-exposed animals rescued by diazepam indicated no additional seizurefor up to 1 h post-TETS exposure.

FIG. 12. Adult male NIH Swiss mice were injected with TETS (i.p.). Twominutes following the second clonic seizure, mice were injected i.p.with diazepam (in saline) or allopregnanolone (AlloP, in β-cyclodextrin)singly or in combination. Seizure time to onset, number and durationwere monitored for 1 h post-TETS exposure. % Survival is at 24 h postTETS injection. Data presented as the mean±SEM (n=6-8 per group).**p<0.05 as determined by one way ANOVA with Tukey's post hoc test.

FIG. 13. Adult male NIH Swiss mice were injected i.p. with diazepam (insaline) or allopregnanolone (AlloP, in β-cyclodextrin) singly or incombination 10 minutes prior to i.p. injection of TETS. Seizure time toonset, duration and number were monitored for 1 h post-TETS injection. %Survival is at 24 h post TETS injection. Data presented as the mean±SEM(n=8 per group). **p<0.01 as determined by one way ANOVA with Tukey'spost hoc test.

FIG. 14 illustrates the effect of benzodiazepine and neuro-steroidtreatments on blood pressure. Adult male NIH Swiss mice were i.p.injected with diazepam (DZP) or allopregnanolone (AlloP) alone or incombination. Blood pressure (BP) was measured using a tail cuff CODAnon-invasive blood measuring system from Kent Scientific. This systemutilizes volume pressure recording technology to detect changes thatcorrespond to systolic and diastolic BP. Diastolic BP is measured andsystolic BP calculated. BP is measured for 6 days prior to testing toobtain baseline BP and allow animals to acclimate to the chamber.Measurements consisted of 20 cycles of 30 sec each with 10 sec delaybetween each measurement. Data presented as the mean±SEM (n=6 pergroup).

DETAILED DESCRIPTION

1. Introduction

Tetramethylenedisulfotetramine (TETS) is a potent convulsant that isconsidered a chemical threat agent. We characterized TETS as anactivator of spontaneous Ca2· oscillations and electrical burstdischarges in mouse hippocampal neuronal cultures at 13-17 days in vitrousing FLIPR® Fluo-4 fluorescence measurements and extracellularmultielectrode array (MEA) recording Acute exposure to TETS (≥2 μM)reversibly altered the pattern of spontaneous neuronal discharges,producing clustered burst firing and an overall increase in dischargefrequency TETS also dramatically affected Ca2+ dynamics causing animmediate but transient elevation of neuronal intracellular Ca2−followed by decreased frequency of Ca2| oscillations having greater peakamplitudes. The effect on Ca2| dynamics was similar to that elicited bypicrotoxin and bicuculline, supporting the view that TETS acts byinhibiting GABA_(A) receptor function. The effect of TETS on Ca2|dynamics requires activation of NMDA receptors, since the changesinduced by TETS were prevented by MK-801 block of NMDA receptors, butnot nifedipine block of L-type Ca2− channels. Pre-treatment with theGABA_(A) receptor positive modulators diazepam and allopregnanolonepartially mitigated TETS-induced changes in Ca2− dynamics. Moreover,low, minimally effective concentrations of diazepam (0.1 μM) andallopregnanolone (0.1 μM), when administered together, were highlyeffective in suppressing TETS-induced alterations in Ca2· dynamics,suggesting that the combination of positive modulators synaptic andextrasynaptic GABA_(A) receptors have therapeutic potential. These rapidthroughput in vitro assays may assist in the identification of singleagents or combinations that have utility in the treatment of TETSintoxication.

2. Conditions Amenable to Treatment

Co-administration of a benzodiazepine and a neurosteroid. In varyingembodiments, one or both of the benzodiazepine and the neurosteroid areadministered in a sub-therapeutic dose or amount finds use in the rapidamelioration and/or termination of seizures. In various embodiments, theseizures may be due to an epileptic condition. Optionally, an NMDAreceptor antagonist is also co-administered.

The term “epilepsy” refers to a chronic neurological disordercharacterized by recurrent unprovoked seizures. These seizures aretransient signs and/or symptoms of abnormal, excessive or synchronousneuronal activity in the brain. There are over 40 different types ofepilepsy, including without limitation childhood absence epilepsy,juvenile absence epilepsy, benign Rolandic epilepsy, clonic seizures,complex partial seizures, frontal lobe epilepsy, febrile seizures,infantile spasms, juvenile myoclonic epilepsy, Lennox-Gastaut syndrome,Landau-Kleffner Syndrome, myoclonic seizures, mitochondrial disordersassociated with seizures, Lafora Disease, progressive myoclonicepilepsies, reflex epilepsy, and Rasmussen's syndrome. There are alsonumerous types of seizures including simple partial seizures, complexpartial seizures, generalized seizures, secondarily generalizedseizures, temporal lobe seizures, tonic-clonic seizures, tonic seizures,psychomotor seizures, limbic seizures, status epilepticus, refractorystatus epilepticus or super-refractory status epilepticus, abdominalseizures, akinetic seizures, autonomic seizures, massive bilateralmyoclonus, drop seizures, focal seizures, gelastic seizures, Jacksonianmarch, motor seizures, multifocal seizures, neonatal seizures, nocturnalseizures, photosensitive seizure, sensory seizures, sylvan seizures,withdrawal seizures and visual reflex seizures.

The most widespread classification of the epilepsies divides epilepsysyndromes by location or distribution of seizures (as revealed by theappearance of the seizures and by EEG) and by cause. Syndromes aredivided into localization-related epilepsies, generalized epilepsies, orepilepsies of unknown localization. Localization-related epilepsies,sometimes termed partial or focal epilepsies, arise from an epilepticfocus, a small portion of the brain that serves as the irritant drivingthe epileptic response. Generalized epilepsies, in contrast, arise frommany independent foci (multifocal epilepsies) or from epileptic circuitsthat involve the whole brain. Epilepsies of unknown localization remainunclear whether they arise from a portion of the brain or from morewidespread circuits.

Epilepsy syndromes are further divided by presumptive cause idiopathic,symptomatic, and cryptogenic. Idiopathic epilepsies are generallythought to arise from genetic abnormalities that lead to alterations inbrain excitability. Symptomatic epilepsies arise from the effects of anepileptic lesion, whether that lesion is focal, such as a tumor, or adefect in metabolism causing widespread injury to the brain. Cryptogenicepilepsies involve a presumptive lesion that is otherwise difficult orimpossible to uncover during evaluation. Forms of epilepsy are wellcharacterized and reviewed, e.g. in Epilepsy: A Comprehensive Textbook(3-volume set), Engel, et al., editors, 2nd Edition, 2007, Lippincott,Williams and Wilkins; and The Treatment of Epilepsy: Principles andPractice, Wyllie, et al., editors, 4th Edition, 2005, Lippincott,Williams and Wilkins; and Browne and Holmes, Handbook of Epilepsy, 4thEdition, 2008, Lippincott, Williams and Wilkins.

3. Subjects Amenable to Treatment

In various embodiments, the patient may be experiencing anelectrographic or behavioral seizure or may be experiencing a seizureaura, which itself is a localized seizure that may spread and become afull blown behavioral seizure. For example, the subject may beexperiencing aura that alerts of the impending onset of a seizure orseizure cluster.

Alternatively, the subject may be using a seizure prediction device thatalerts of the impending onset of a seizure or seizure cluster.Implantable seizure prediction devices are known in the art anddescribed, e.g., in D'Alessandro, et al., IEEE TRANSACTIONS ONBIOMEDICAL ENGINEERING, VOL. 50. NO. 5. MAY 2003, and U.S. PatentPublication Nos. 2010/0198098, 2010/0168603, 2009/0062682, and2008/0243022.

The subject may have a personal or familial history of any of theepileptic conditions described herein. The subject may have beendiagnosed as having any of the epileptic conditions described herein. Insome embodiments, the subject has or is at risk of suffering statusepilepticus, refractory status epilepticus or super-refractory statusepilepticus. In some embodiments, the subject has or is at risk ofsuffering a myoclonic seizure or myoclonic epilepsy, e.g., juvenilemyoclonic epilepsy. The PTZ seizure model demonstrated herein ispredictive of utility and/or activity in counteracting myoclonicseizures or myoclonic epilepsy in humans.

In various embodiments, the subject may be at risk of exposure to or mayhave been exposed to tetramethylenedisulfotetramine (TETS).

In various embodiments, the subject may be at risk of exposure to or mayhave been exposed to a nerve agent or a pesticide that can causeseizures. Illustrative nerve agents that can cause seizures include,e.g., organophosphorus nerve agents, e.g., tabun, sarin, soman, GF, VRand/or VX. Illustrative pesticides that can cause seizures include,e.g., organophosphate pesticides (e.g., Acephate (Orthene),Azinphos-methyl (Gusathion, Guthion), Bensulide (Betasan, Lescosan),Bomyl (Swat), Bromophos (Nexion), Bromophos-ethyl (Nexagan), Cadusafos(Apache, Ebufos, Rugby), Carbophenothion (Trithion), Chlorethoxyfos(Fortress), Chlorfenvinphos (Apachlor, Birlane), Chlormephos (Dotan),Chlorphoxim (Baythion-C), Chlorpyrifos (Brodan, Dursban, Lorsban),Chlorthiophos (Celathion), Coumaphos (Asuntol, Co-Ral), Crotoxyphos(Ciodrin, Cypona), Crufomate (Ruelene), Cyanofenphos (Surecide),Cyanophos (Cyanox), Cythioate (Cyflee, Proban), DEF (De-Green), E-Z-OffD), Demeton (Systox), Demeton-S-methyl (Duratox, Metasystoxl), Dialifor(Torak), Diazinon, Dichlorofenthion, (VC-13 Nemacide), Dichlorvos (DDVP,Vapona), Dicrotophos (Bidrin), Dimefos (Hanane, Pestox XIV), Dimethoate(Cygon, DeFend), Dioxathion (Delnav), Disulfoton (Disyston), Ditalimfos,Edifenphos, Endothion, EPBP (S-seven), EPN, Ethion (Ethanox), Ethoprop(Mocap), Ethyl parathion (E605, Parathion, thiophos), Etrimfos (Ekamet),Famphur (Bash, Bo-Ana, Famfos), Fenamiphos (Nemacur), Fenitrothion(Accothion, Agrothion, Sumithion), Fenophosphon (Agritox,trichloronate), Fensulfothion (Dasanit), Fenthion (Baytex, Entex,Tiguvon), Fonofos (Dyfonate, N-2790), Formothion (Anthio), Fosthietan(Nem-A-Tak), Heptenophos (Hostaquick), Hiometon (Ekatin), Hosalone(Zolone), IBP (Kitazin), Iodofenphos (Nuvanol-N), Isazofos (Brace,Miral, Triumph), Isofenphos (Amaze, Oftanol), Isoxathion (E-48,Karphos), Leptophos (Phosvel), Malathion (Cythion), Mephosfolan(Cytrolane), Merphos (Easy Off-D, Folex), Methamidophos (Monitor),Methidathion (Supracide, Ultracide), Methyl parathion (E601. Penncap-M).Methyl trithion, Mevinphos (Duraphos, Phosdrin), Mipafox (Isopestox,Pestox XV), Monocrotophos (Azodrin), Naled (Dibrome), Oxydemeton-methyl(Metasystox-R), Oxydeprofos (Metasystox-S), Phencapton (G 28029),Phenthoate (Dimephenthoate, Phenthoate), Phorate (Rampart, Thimet),Phosalone (Azofene, Zolone), Phosfolan (Cylan, Cyolane), Phosmet(Imidan, Prolate), Phosphamidon (Dimecron), Phostebupirim (Aztec),Phoxim (Baythion), Pirimiphos-ethyl (Primicid), Pirimiphos-methyl(Actellic), Profenofos (Curacron), Propetamphos (Safrotin), Propylthiopyrophosphate (Aspon), Prothoate (Fac), Pyrazophos (Afugan,Curamil), Pyridaphenthion (Ofunack), Quinalphos (Bayrusil), Ronnel(Fenchlorphos, Korlan), Schradan (OMPA), Sulfotep (Bladafum, Dithione,Thiotepp), Sulprofos (Bolstar, Helothion), Temephos (Abate, Abathion),Terbufos (Contraven, Counter), Tetrachlorvinphos (Gardona, Rabon),Tetraethyl pyrophosphate (TEPP), Triazophos (Hostathion), andTrichlorfon (Dipterex, Dylox, Neguvon, Proxol).

4. Therapeutic Agents

Generally, the compositions and methods comprise co-administering abenzodiazepine and a neurosteroid. In varying embodiments, one or bothof the benzodiazepine and the neurosteroid are co-administered at asub-therapeutic dose or amount. Optionally, an NMDA receptor antagonistis co-administered. The agents can be co-administered concurrently orsequentially. The agents can be co-administered via the same ordifferent routes of administration. In various embodiments, the agentsare co-administered in a single composition.

a. Benzodiazepines

Any benzodiazepine known in the art finds use in the presentcompositions and methods. Illustrative benzodiazepine that find useinclude without limitation bretazenil, clonazepam, cloxazolam,clorazepate, diazepam, fludiazepam, flutoprazepam, lorazepam, midazolam,nimetazepam, nitrazepam, phenazepam, temazepam and clobazam. In someembodiments, the benzodiazepine is midazolam. In some embodiments, thebenzodiazepine is diazepam.

b. Neurosteroids

The terms “neuroactive steroid” or “neurosteroids” interchangeably referto steroids that rapidly alter neuronal excitability through interactionwith neurotransmitter-gated ion channels, specifically GABA_(A)receptors Neuroactive steroids have a wide range of applications fromsedation to treatment of epilepsy and traumatic brain injury.Neuroactive steroids act as direct agonists and allosteric positivemodulators of GABA_(A) receptors. Several synthetic neuroactive steroidshave been used as sedatives for the purpose of general anaesthesia forcarrying out surgical procedures. Exemplary sedating neuroactivesteroids include without limitation alphaxolone, alphadolone,hydroxydione and minaxolone. The neuroactive steroid ganaxolone findsuse for the treatment of epilepsy. In various embodiments, thebenzodiazepine or non-benzodiazepine benzodiazepine receptor agonist isco-administered with an endogenously occurring neurosteroid or otherneuroactive steroid. Illustrative endogenous neuroactive steroids, e.g.,allopregnanolone and tetrahydrodeoxycorticosterone find use. In someembodiments, the neurosteroid is selected from the group consisting ofallopregnanolone, allotetrahydrodeoxycorticosterone, ganaxolone,alphaxolone, alphadolone, hydroxydione, minaxolone, and Althesin.

In various embodiments the neurosteroid is allopregnanolone (ALP)Allopregnanolone, also known as 3α-hydroxy-5α-pregnan-20-one or3α,5α-tetrahydroprogesterone, IUPAC name1-(3-Hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethanone,and referenced as CAS number 516-54-1, is a prototypic neurosteroidpresent in the blood and also the brain. It is a metabolite ofprogesterone and modulator of GABA_(A) receptors. Whileallopregnanolone, like other GABA_(A) receptor active neurosteroids suchas allotetrahydrodeoxycorticosterone (3α,21-dihydroxy-5α-pregnan-20-one;THDOC), positively modulates all GABA_(A) receptor isoforms, thoseisoforms containing δ-subunits exhibit greater magnitude potentiationAllopregnanolone has pharmacological properties similar to otherpositive modulators of GABA_(A) receptors, including anxiolytic andanticonvulsant activity. Allopregnanolone is neuroprotective in manyanimal models of neurodegenerative conditions, including, e.g.,Alzheimer's disease (Wang et al, Proc Natl Acad Sci USA. 2010 Apr. 6;107(14):6498-503), cerebral edema (Limmroth et al., Br J Pharmacol. 1996January; 117(1):99-104) and traumatic brain injury (He et al., RestorNeurol Neurosci. 2004; 22(1):19-31; and He, et al., Exp Nerol. 2004October; 189(2):404-12), Mood disorders (Robichaud and Debonnel, Int JNeuropsychopharmacol. 2006 April; 9(2): 191-200), Niemann-Pick type Cdisease (Griffin et al., Nat Med. 2004 July, 10(7) 704-11) and acts asan anticonvulsant against chemically induced seizures, including thepentylentetrazol (PTZ) model (Kokate et al., J Pharmacol Exp Ther. 1994September; 270(3) 1223-9) The chemical structure of allopregnanolone isdepicted below in Formula I:

In various embodiments, the compositions comprise a sulfate, salt,hemisuccinate, nitrosylated, derivative or congener of allopregnanolone.

Delivery of other neurosteroids also can be enhanced by formulation in acyclodextrin and/or in an edible oil. Other neurosteroids that can beformulated in a cyclodextrin and/or in an edible oil, include withoutlimitation allotetrahydrodeoxycorticosterone(3α,21-dihydroxy-5α-pregnan-20-one; THDOC), 3α,21-dihydroxy-5b-pregnan-20-one, pregnanolone(3α-hydroxy-5β-pregnan-20-one), Ganaxolone (INN, also known as CCD-1042;IUPAC name (3α,5α)-3-hydroxy-5-methylpregnan-20-one;1-[(3R,5S,8R,9S,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethyl-1,2,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydrocyclopenta[a]phenanthren-17-yl]ethanone),alphaxolone, alphadolone, hydroxydione, minaxolone, and Althesin (amixture of alphaxolone, alphadolone, tetrahydrodeoxycorticosterone,pregnenolone, dehydroepiandrosterone (DHEA), 7-substitutedbenz[c]indene-3-carbonitriles (see, e.g., Hu, et al., J Med Chem. (1993)36(24):3956-67); 7-(2-hydroxyethyl)benz[e]indene analogues (see, e.g.,Han, et al., J Med Chem. (1995) 38(22):4548-56); 3 alpha-hydroxy-5alpha-pregnan-20-one and 3 alpha-hydroxy-5 beta-pregnan-20-one analogues(see. e.g., Han, et al., J Med Chem. (1996) 39(21):4218-32); enantiomersof dehydroepiandrosterone sulfate, pregnenolone sulfate, and(3alpha,5beta)-3-hydroxypregnan-20-one sulfate (see, e.g., Nilsson, etal., J Med Chem. (1998) 41(14):2604-13): 13,24-cyclo-18,21-dinorcholaneanalogues (see, e.g., Jiang, et al, J Med Chem. (2003) 46(25):5334-48):N-acylated 17a-aza-D-homosteroid analogues (see, e.g., Covey, et al, JMed Chem. (2000) 43(17):3201-4); 5 beta-methyl-3-ketosteroid analogues(see, e.g., Zeng, et al., J Org Chem. (2000) 65(7):2264-6);18-norandrostan-17-one analogues (see, e.g., Jiang, et al., J Org Chem.(2000) 65(11):3555-7); (3alpha,5alpha)- and(3alpha,5beta)-3-hydroxypregnan-20-one analogs (see. e.g., Zeng, et al.,J Med Chem. (2005) 48(8):3051-9); benz[f]indenes (see, e.g., Scaglione,et al., J Med Chem. (2006) 49(15):4595-605); enantiomers of androgens(see, e.g., Katona, et al., Eur J Med Chem. (2008) 43(1): 107-13),cyclopenta[b]phenanthrenes and cyclopenta[b]anthracenes (see, e.g.,Scaglione, et al., J Med Chem. (2008) 51(5) 1309-18),2beta-hydroxygonane derivatives (see, e.g., Wang, et al., Tetrahedron(2007) 63(33):7977-7984), Δ16-alphaxalone and corresponding17-carbonitrile analogues (see, e.g., Bandyopadhyaya, et al., Bioorg MedChem Lett. (2010) 20(22):6680-4); A(16) and A(17(20)) analogues ofA(16)-alphaxalone (see, e.g., Stastna, et al., J Med Chem. (2011)54(11):3926-34); neurosteroid analogs developed by CoCensys (now PurdueNeuroscience) (e.g., CCD-3693, Co2-6749 (a.k.a., GMA-839 andWAY-141839); neurosteroid analogs described in U.S. Pat. No. 7,781,421and in PCT Patent Publications WO 2008/157460; WO 1993/003732; WO1993/018053; WO 1994/027608; WO 1995/021617; WO 1996/016076; WO1996/040043, as well as salts, hemisuccinates, nitrosylated, sulfatesand derivatives thereof.

In various embodiments, the steroid or neurosteroid is not a sexhormone. In various embodiments, the steroid or neurosteroid is notprogesterone.

As appropriate, the steroid or neurosteroid (e.g., allopregnanolone) mayor may not be micronized. As appropriate, the steroid or neurosteroid(e.g., allopregnanolone) may or may not be enclosed in microspheres insuspension in the oil.

c. NMDA Receptor Antagonists

Illustrative NMDA receptor antagonists that find use include withoutlimitation, e.g., dizocilpine (MK-801), meperidine, methadone,dextropropoxyphene, tramadol, ketobemidone, ketamine, dextromethorphan,phencyclidine, nitrous oxide (N₂O), AP5(R-2-amino-5-phosphonopentanoate), AP7 (2-amino-7-phosphonoheptanoicacid), CPPene (3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonicacid), selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,eticyclidine, gacyclidine, ibogaine, magnesium, memantine,methoxetamine, rolicyclidine.tenocyclidine, methoxydine, tiletamine,xenon, neramexane, eliprodil, etoxadrol, dexoxadrol, WMS 2539, NEFA,remacemide, delucemine, 8A-PDHQ, aptiganel, 1 HU-211, rhynchophylline,1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA(5,7-dichlorokynurenic acid), kynurenic acid, lacosamide, CP-101,606(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some embodiments,the NMDA receptor antagonist is selected from the group consisting ofketamine, dextromethorphan, phencyclidine, CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine, dextrorphan, memantine, tiletamine, neramexane,cliprodil, remacemide, aptiganel, 1-Aminocyclopropanecarboxylic acid(ACPC), 7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenicacid, CP-101,606 (traxoprodil), AZD6765 (lanicemine) and GLYX-13. Insome embodiments, the NMDA receptor antagonist is dizocilpine (MK-801).

5. Formulation and Administration

In various embodiments, one or more of the benzodiazepines and one ormore neurosteroids are formulated for intramuscular, intravenous,subcutaneous, intrapulmonary and/or inhalational administration. Invarious embodiments, the benzodiazepines are formulated for delivery viaan inhaler. In various embodiments other routes of delivery, describedherein may be appropriate. Optionally a NMDA receptor antagonist isincluded in the compositions and/or co-administration.

Appropriate dosing will depend on the sire and health of the patient andcan be readily determined by a trained clinician. Initial doses are lowand then can be incrementally increased until the desired therapeuticeffect is achieved with little or no adverse side effects. Determinationof an effective amount for administration in a single dosage is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein Generally, an efficacious oreffective amount of the agents (e.g., one or more benzodiazepines andone or more neurosteroids, optionally including a NMDA receptorantagonist) is determined by first administering a low dose or smallamount of the agent and then incrementally increasing the administereddose or dosages, adding a second or third medication as needed, until adesired effect of is observed in the treated subject with minimal or notoxic side effects. Applicable methods for determining an appropriatedose and dosing schedule for administration of a combination of agentsof the present invention are described, for example, in Goodman andGilman's The Pharmacological Basis of Therapeutics, 12th Edition, 2010,supra, in a Physicians' Desk Reference (PDR), 67^(th) Edition, 2013; inRemington: The Science and Practice of Pharmacy, 21^(st) Ed., 2005,supra, and in Martindale: The Complete Drug Reference, Sweetman, 2005,London: Pharmaceutical Press., and in Martindale, Martindale: The ExtraPharmacopoeia, 31st Edition, 1996, Amer Pharmaceutical Assn, each ofwhich are hereby incorporated herein by reference.

In various embodiments, the agents (e.g., one or more benzodiazepinesand one or more neurosteroids, optionally including a NMDA receptorantagonist) are nebulized. Methods and systems for intrapulmonarydelivery of agents, e.g., benzodiazepines, are known in the art and finduse. Illustrative systems for aerosol delivery of benzodiazepines byinhalation are described, e.g., in U.S. Pat. Nos. 5,497,763; 5,660,166;7,060,255; and 7,540,286; and U.S. Patent Publication Nos. 2003/0032638;and 2006/0052428, each of which are hereby incorporated herein byreference in their entirety for all purposes. Preferably, the agents(e.g., one or more benzodiazepines and one or more neurosteroids,optionally including a NMDA receptor antagonist) are nebulized withoutthe input of heat.

For administration of the nebulized and/or aerosolized agents (e.g., oneor more benzodiazepines and one or more neurosteroids, optionallyincluding a NMDA receptor antagonist), the size of the aerosolparticulates can be within a range appropriate for intrapulmonarydelivery, particularly delivery to the distal alveoli. In variousembodiments, the aerosol particulates have a mass median aerodynamicdiameter (“MMAD”) of less than about 5 μm, 4 μm, 3 μm, for example,ranging from about 1 μm to about 3 μm, e.g., from about 2 μm to about 3μm, e.g., ranging from about 0.01 μm to about 0.10 μm. Aerosolscharacterized by a MMAD ranging from about 1 μm to about 3 μm candeposit on alveoli walls through gravitational settling and can beabsorbed into the systemic circulation, while aerosols characterized bya MMAD) ranging from about 0.01 μm to 0.10 μm can also be deposited onthe alveoli walls through diffusion. Aerosols characterized by a MMAD)ranging from about 0.15 μm to about 1 μm are generally exhaled. Thus, invarious embodiments, aerosol particulates can have a MMAD ranging from0.01 μm to about 5 μm, for example, ranging from about 0.05 μm to about3 μm, for example, ranging from about 1 μm to about 3 μm, for example,ranging from about 0.01 μm to about 0.1 μm. The nebulized and/oraerosolized benzodiazepines can be delivered to the distal alveoli,allowing for rapid absorption and efficacy.

In various embodiments, the agents (e.g., one or more benzodiazepinesand one or more neurosteroids, optionally including a NMDA receptorantagonist) are formulated in a solution comprising excipients suitablefor aerosolized intrapulmonary delivery. The solution can comprise oneor more pharmaceutically acceptable carriers and/or excipients.Pharmaceutically acceptable refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S Pharmacopoeia or other generally recognized pharmacopoeia for use inanimals, and more particularly in humans. Preferably, the solution isbuffered such that the solution is in a relatively neutral pH range, forexample, a pH in the range of about 4 to 8, for example, a pH in therange of about 5-7. In some embodiments, the benzodiazepine isformulated in a buffered solution, for example, phosphate-bufferedsaline.

In various embodiments, the agents (e.g., one or more benzodiazepinesand one or more neurosteroids, optionally including a NMDA receptorantagonist) are prepared as a concentrated aqueous solution. Ordinarymetered dose liquid inhalers have poor efficiency for the delivery tothe deep lung because the particle size is not sufficiently small (Kimet al. 1985 Am Rev Resp Dis 132: 137-142; and Farr et al., 1995 Thorax50:639-644) These systems are therefore used mostly for local deliveryof drugs to the pulmonary airways. In addition, metered doses inhalersmay not be able to deliver sufficient volumes of even a concentratedmidazolam solution to produce the desired rapid antiseizure effect.Accordingly, in various embodiments, a metered doses inhaler is not usedfor delivery of the benzodiazepine, e.g., midazolam. In one embodiment anebulization system with the capability of delivering <5 μm particles(e.g., the PARI LC Star, which has a high efficiency, 78% respirablefraction 0.1-5 μm, see, e.g., pari.com) is used for intrapulmonaryadministration. Electronic nebulizers which employ a vibrating mesh oraperture plate to generate an aerosol with the required particle sizecan deliver sufficient quantities rapidly and find use (See, e.g., Knochand Keller, 2005 Expert Opin Drug Deliv 2: 377-390). Also,custom-designed hand-held, electronic nebulizers can be made and finduse.

Aerosolized delivery of the agents (e.g., one or more benzodiazepinesand one or more neurosteroids, optionally including a NMDA receptorantagonist) can allow for reduced dosing to achieve desired efficacy,e.g., in comparison to intravenous or intranasal delivery.

In various embodiments, the agents (e.g., one or more benzodiazepinesand one or more neurosteroids, optionally including a NMDA receptorantagonist) are dissolved or suspended in a cyclodextrin. In varyingembodiments, the cyclodextrin is an α-cyclodextrin, a β-cyclodextrin ora γ-cyclodextrin. In varying embodiments, the cyclodextrin is selectedfrom the group consisting of hydroxypropyl-β-cyclodextrin, endotoxincontrolled β-cyclodextrin sulfobutyl ethers, or cyclodextrin sodiumsalts (e.g., CAPTTISOL®). Such formulations are useful forintramuscular, intravenous and/or subcutaneous administration.

In various embodiments, the agents (e.g., one or more benzodiazepinesand one or more neurosteroids, optionally including a NMDA receptorantagonist) are dissolved or suspended in an oil that is edible and/ordigestible by the subject, e.g., without undesirable side effects.

In various embodiments, the edible oil comprises one or more vegetableoils. In various embodiments, the vegetable oil is selected from thegroup consisting of coconut oil, corn oil, cottonseed oil, olive oil,palm oil, peanut oil, rapeseed oil, canola oil, safflower oil, sesameoil, soybean oil, sunflower oil, and mixtures thereof.

In some embodiments, the edible oil comprises one or more nut oils. Insome embodiments, the nut oil is selected from the group consisting ofalmond oil, cashew oil, hazelnut oil, macadamia oil, mongongo nut oil,pecan oil, pine nut oil, pistachio oil, walnut oil, and mixturesthereof.

In some embodiments, the edible oil does not comprise castor oil. Insome embodiments, the edible oil does not comprise peanut oil.

Generally, the oils used in the present compositions are isolated fromthe source, e.g., plant, and used without including further additives(e.g., surfactants, acids (organic or fatty), alcohols, esters,co-solvents, solubilizers, lipids, polymers, glycols) or processing. Invarious embodiments, the oil vehicle further comprises a preservative(e.g., vitamin E).

The oil-agents (e.g., one or more benzodiazepines and one or moreneurosteroids, optionally including a NMDA receptor antagonist)compositions can be formulated for oral and/or transmucosal deliveryusing any method known in the art. In one embodiment, the oil-agents(e.g., one or more benzodiazepines and one or more neurosteroids,optionally including a NMDA receptor antagonist) composition isformulated in a capsule, e.g., for oral delivery.

a. Capsules

The capsule shells can be prepared using one or more film formingpolymers. Suitable film forming polymers include natural polymers, suchas gelatin, and synthetic film forming polymers, such as modifiedcelluloses. Suitable modified celluloses include, but are not limitedto, hydroxypropyl methyl cellulose, methyl cellulose, hydroxypropylmethyl cellulose acetate succinate, hydroxypropyl methyl cellulosephthalate, and cellulose acetate phthalate. Hard or soft capsules can beused to administer the hormone. Hard shell capsules are typicallyprepared by forming the two capsule halves, filling one of the halveswith the fill solution, and then sealing the capsule halves together toform the finished capsule. Soft gelatin capsules are typically preparedusing a rotary die encapsulation process as described below.

i. Gelatin Capsules

Gelatin is the product of the partial hydrolysis of collagen. Gelatin isclassified as either Type A or Type B gelatin. Type A gelatin is derivedfrom the acid hydrolysis of collagen while Type B gelatin is derivedfrom the alkaline hydrolysis of collagen. Traditionally, bovine bonesand skins have been used as raw materials for manufacturing Type A andType B gelatin while porcine skins have been used extensively formanufacturing Type A gelatin. In general, acid-processed gelatins formstronger gels than lime-processed gelatins of the same average molecularweight. The capsules can be formulated as hard or soft gelatin capsules.

ii. Non-Gelatin Capsules

Capsules can be prepared from non-gelatin materials, such as carrageenanor modified celluloses. Carrageenan is a natural polysaccharidehydrocolloid, which is derived from seaweed. It includes a linearcarbohydrate polymer of repeating sugar units, without a significantdegree of substitution or branching. Most, if not all, of the galactoseunits on a carrageenan molecule possess a sulfate ester group. There arethree main types of carrageenan: kappa, iota and lambda; although minorforms called mu and nu carrageenan also exist.

iii. Shell Additives

Suitable shell additives include plasticizers, opacifiers, colorants,humectants, preservatives, flavorings, and buffering salts and acids,and combinations thereof.

Plasticizers are chemical agents added to gelatin to make the materialsofter and more flexible. Suitable plasticizers include, but are notlimited to, glycerin, sorbitol solutions which are mixtures of sorbitoland sorbitan, and other polyhydric alcohols such as propylene glycol andmaltitol or combinations thereof.

Opacifiers are used to opacify the capsule shell when the encapsulatedactive agents are light sensitive. Suitable opacifiers include titaniumdioxide, zinc oxide, calcium carbonate and combinations thereof.

Colorants can be used for marketing and productidentification/differentiation purposes. Suitable colorants includesynthetic and natural dyes and combinations thereof.

Humectants can be used to suppress the water activity of the softgel.Suitable humectants include glycerin and sorbitol, which are oftencomponents of the plasticizer composition. Due to the low water activityof dried, properly stored softgels, the greatest risk frommicroorganisms comes from molds and yeasts. For this reason,preservatives can be incorporated into the capsule shell. Suitablepreservatives include alkyl esters of p-hydroxy benzoic acid such asmethyl, ethyl, propyl, butyl and heptyl esters (collectively known as“parabens”) or combinations thereof.

Flavorings can be used to mask unpleasant odors and tastes of fillformulations. Suitable flavorings include synthetic and naturalflavorings. The use of flavorings can be problematic due to the presenceof aldehydes which can cross-link gelatin. As a result, buffering saltsand acids can be used in conjunction with flavorings that containaldehydes in order to inhibit cross-linking of the gelatin.

b. Enteric Capsules

Alternatively, the liquid fills can be incorporated into an entericcapsule, wherein the enteric polymer is a component of the capsuleshell, as described in WO 2004/030658 to Banner Pharmacaps, Inc. Theenteric capsule shell is prepared from a mass comprising a film-formingpolymer, an acid-insoluble polymer which is present in an amount makingthe capsule resistant to the acid within the stomach, an aqueoussolvent, and optionally, one or more plasticizers and/or colorants.Other suitable shell additives including opacifiers, colorants,humectants, preservatives, flavorings, and buffering salts and acids maybe added.

i. Film-Forming Polymers

Exemplary film-forming polymers can be of natural or synthetic origin.Natural film-forming polymers include gelatin and gelatin-like polymers.Other suitable natural film-forming polymers include shellac, alginates,pectin, and zeins. Synthetic film-forming polymers include hydroxypropylmethyl cellulose, methyl cellulose, hydroxypropyl methyl celluloseacetate succinate, hydroxypropyl methyl cellulose phthalate, celluloseacetate phthalate, and acrylates such as poly (meth)acrylate. The weightratio of acid-insoluble polymer to film-forming polymer is from about15% to about 50% In one embodiment, the film forming polymer is gelatin.

ii. Acid-Insoluble Polymers

Exemplary acid-insoluble polymers include cellulose acetate phthalate,cellulose acetate butyrate, hydroxypropyl methyl cellulose phthalate,algenic acid salts such as sodium or potassium alginate, shellac,pectin, acrylic acid-methylacrylic acid copolymers (available under thetradename EUDRAGIT® from Rohm America Inc., Piscataway, N.J. as a powderor a 30% aqueous dispersion; or under the tradename EASTACRYL®, fromEastman Chemical Co. Kingsport. Tenn., as a 30% dispersion) in oneembodiment, the acid-insoluble polymer is EUDRAGIT® L100, which is amethacrylic acid/methacrylic acid methyl ester copolymer. Theacid-insoluble polymer is present in an amount from about 8% to about20% by weight of the wet gelatin mass. The weight ratio ofacid-insoluble polymer to film-forming polymer is from about 15% toabout 50%.

iii. Aqueous Solvent

Hard and soft capsules are typically prepared from solutions orsuspensions of the film forming polymer and the acid-insoluble polymer.Suitable solvents include water, aqueous solvents, and organic solvents.In one embodiment, the solvent is water or an aqueous solvent. Exemplaryaqueous solvents include water or aqueous solutions of alkalis such asammonia, sodium hydroxide, potassium hydroxide, ethylene diamine,hydroxylamine, tri-ethanol amine, or hydroalcoholic solutions of thesame. The alkali can be adjusted such that the final pH of the gelatinmass is less than or equal to 9.0, preferably less than or equal to 8.5,more preferably less than or equal to 8.0. In one embodiment, the alkaliis a volatile alkali such as ammonia or ethylene diamine. Upon drying ofthe finished capsule, the water content of the capsule is from about 2%to about 10% by weight of the capsule, preferably from about 4% to about8% by weight of the capsule.

iv. Plasticizers

Exemplary plasticizers include glycerol, glycerin, sorbitol,polyethylene glycol, citric acid, citric acid esters such astriethylcitrate, polyalcohols with 3-6 carbons and combinations thereof.The plasticizer to polymer (film forming polymer plus acid-insolublepolymer) ratio is from about 10% to about 50% b of the polymer weight.

c. Methods of Manufacture

i. Capsule Fill

The fill material is prepared by dissolving the steroid or neurosteroid(e.g., allopregnanolone) in the carrier containing a fatty acid solvent,such as oleic acid. The mixture of hormone and fatty acid may be heatedto facilitate dissolution of the hormone. Upon cooling to roomtemperature and encapsulation, the solution remains a liquid. The fillis typically deacrated prior to encapsulation in a soft gelatin capsule.Additional excipients including, but not limited to, co-solvents,antioxidants may be added to the mixture of the hormone and fatty acidAgain the mixture may be heated to facilitate dissolution of theexcipients. The steroid or neurosteroid (e.g., allopregnanolone) isfully dissolved in the carrier of the present invention and remains soupon storage.

ii. Capsule Shell

a. Gelatin or Non-Gelatin Capsules

The main ingredients of the capsule shell are gelatin (or a gelatinsubstitute for non-gelatin capsules), plasticizer, and purified water.The primary difference between soft and hard capsules is the amount ofplasticizer present in the capsule shell.

Typical gel formulations contain (w/w) 40-50% gelatin, 20-30%plasticizer, and 30-40% purified water. Most of the water issubsequently lost during capsule drying. The ingredients are combined toform a molten gelatin mass using either a cold melt or a hot meltprocess. The prepared gel masses are transferred to preheated,temperature-controlled, jacketed holding tanks where the gel mass isaged at 50-60° C. until used for encapsulation.

i. Cold Melt Process

The cold melt process involves mixing gelatin with plasticizer andchilled water and then transferring the mixture to a jacket-heated tank.Typically, gelatin is added to the plasticizer at ambient temperature(18-22° C.). The mixture is cooked (57-95° C.) under vacuum for 15-30minutes to a homogeneous, deaerated gel mass. Additional shell additivescan be added to the gel mass at any point during the gel manufacturingprocess or they may be incorporated into the finished gel mass using ahigh torque mixer.

ii. Hot Melt Process

The hot melt process involves adding, under mild agitation, the gelatinto a preheated (60-80° C.) mixture of plasticizer and water and stirringthe blend until complete melting is achieved. While the hot melt processis faster than the cold melt process, it is less accurately controlledand more susceptible to foaming and dusting.

h. Soft Capsules

Soft capsules are typically produced using a rotary die encapsulationprocess. The gel mass is fed either by gravity or through positivedisplacement pumping to two heated (48-65° C.) metering devices. Themetering devices control the flow of gel into cooled (10-18° C.),rotating casting drums. Ribbons are formed as the cast gel masses set oncontact with the surface of the drums.

The ribbons are fed through a series of guide rolls and betweeninjection wedges and the capsule-forming dies A food-grade lubricant oilis applied onto the ribbons to reduce their tackiness and facilitatetheir transfer. Suitable lubricants include mineral oil, medium chaintriglycerides, and soybean oil. Fill formulations are fed into theencapsulation machine by gravity. In the preferred embodiment, the softcapsules contain printing on the surface, optionally identifying theencapsulated agent and/or dosage.

Upon drying of the finished capsule, the water content of the capsule isfrom about 2% to about 10% by weight of the capsule, preferably fromabout 4% to about 8% by weight of the capsule.

c. Enteric Capsules

A method of making an enteric capsule shell is described in WO2004/030658 to Banner Pharmacaps, Inc. The enteric mass is typicallymanufactured by preparing an aqueous solution comprising a film-forming,water soluble polymer and an acid-insoluble polymer and mixing thesolution with one or more appropriate plasticizers to form a gelatinmass. Alternatively, the enteric mass can be prepared by using aready-made aqueous dispersion of the acid-insoluble polymer by addingalkaline materials such as ammonium, sodium, or potassium hydroxides orother alkalis that will cause the acid-insoluble polymer to dissolve.The plasticizer-wetted, film-forming polymer can then be mixed with thesolution of the acid-insoluble polymer. The mass can also be prepared bydissolving the acid-insoluble polymer or polymers in the form of saltsof the above-mentioned bases or alkalis directly in water and mixing thesolution with the plasticizer-wetted, film-forming polymer. The mass iscast into films or ribbons using heat controlled drums or surfaces. Thefill material is encapsulated in a soft capsule using a rotary die. Thecapsules are dried under controlled conditions of temperature andhumidity. The final moisture content of the shell composition is fromabout 2% to about 10% by weight of the capsule shell, preferably fromabout 4% to about 8% by weight by weight of the capsule shell.

Alternatively, release of the agents (e.g., one or more benzodiazepinesand one or more neurosteroids, optionally including a NMDA receptorantagonist) from the capsule can be modified by coating the capsule withone or more modified release coatings, such as sustained releasecoatings, delayed release coatings, and combinations thereof.

The concentration of the agents (e.g., one or more benzodiazepines andone or more neurosteroids, optionally including a NMDA receptorantagonist) in the vehicle (e.g., cyclodextrin and/or edible oil) ispreferably in unit dosage form. The term “unit dosage form”, as used inthe specification, refers to physically discrete units suitable asunitary dosages for human subjects and animals, each unit containing apredetermined quantity of active material calculated to produce thedesired pharmaceutical effect in association with the requiredpharmaceutical diluent, carrier or vehicle. The specifications for thenovel unit dosage forms of this invention are dictated by and directlydependent on (a) the unique characteristics of the active material andthe particular effect to be achieved and (b) the limitations inherent inthe art of compounding such an active material for use in humans andanimals, as disclosed in detail in this specification, these beingfeatures of the present invention.

In various embodiments, the benzodiazepines are administered at a dosethat is less than about 10%, 15%, 25%, 50% or 75% of established dosesfor their administration for the prevention or mitigation of anepileptic seizure. In some embodiments, the benzodiazepine isadministered at a dose in the range of about 0.05 mg/kg to about 1.0mg/kg, for example, about 0.2 mg/kg to about 0.8 mg/kg, for example,about 0.05 mg/kg, 0.08 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, or 1.0mg/kg. In some embodiments the benzodiazepine is administered at a dosein the range of about 10 μg/kg to about 80 μg/kg, for example, about 20μg/kg to about 60 μg/kg, for example, about 25 g/kg to about 50 g/kg,for example, about 10 μg/kg, 15 μg/kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35μg/kg, 40 μg/kg, 45 μg/kg, 50 μg/kg, 60 μg/kg, 70 μg/kg, or 80 μg/kg. Insome embodiments, the benzodiazepine is administered at a dose in therange of about 0.3 μg/kg to about 3.0 μg/kg. In varying embodiments, thebenzodiazepine is administered at a dose that does not decrease bloodpressure. When co-administered with one or more neurosteroids, thebenzodiazepine can be co-administered at a dose that is less than about10%, 15%, 259%, 50% or 75% of the aforementioned doses or at a dose thatis less than about 10%, 15%, 25%, 50% or 75% of established doses fortheir administration for the prevention or mitigation of an epilepticseizure. When co-administered with one or more neurosteroids, thebenzodiazepine can be co-administered at a dose that is less than about10%, 15%, 25%, 50% or 75% of doses known to be efficacious via aselected route of administration (e.g., oral, intramuscular,intravenous, subcutaneous and/or intrapulmonary).

In various embodiments, the compositions are formulated foradministration of about 5 mg/kg to about 50 mg/kg of the steroid orneurosteroid (e.g., allopregnanolone), e.g., about 5 mg/kg, 10 mg/kg, 15mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50mg/kg. When co-administered with one or more benzodiazepines, thesteroid or neurosteroid (e.g., allopregnanolone) can be co-administeredat a dose that is less than about 10%, 15%, 259%, 50% or 75% of theaforementioned doses or at a dose that is less than about 10%, 15%, 25%,50% or 75% of established doses for their administration for theprevention or mitigation of an epileptic seizure. When co-administeredwith one or more benzodiazepines, the neurosteroid can beco-administered at a dose that is less than about 10%, 15%, 25%, 50% or75% of doses known to be efficacious via a selected route ofadministration (e.g., oral, intramuscular, intravenous, subcutaneousand/or intrapulmonary).

6. Monitoring Efficacy

Co-administration of a benzodiazepine and a neurosteroid (optionallywith an NMDA receptor antagonist) to a subject results in the preventionof the occurrence of an impending seizure and/or the rapid terminationor abortion of a seizure in progress.

In various embodiments, efficacy can be monitored by the subject. Forexample, in a subject experiencing aura or receiving a warning from aseizure prediction device, the subject can self-administer via theintrapulmonary route a dose of the benzodiazepine. If the benzodiazepineis administered in an efficacious amount, the sensation of aura shouldsubside and/or the seizure prediction device should no longer predictthe imminent occurrence of an impending seizure. If the sensation ofaura does not subside and/or the seizure prediction device continues topredict an impending seizure, a second dose of benzodiazepine can beadministered.

In other embodiments, the efficacy is monitored by a caregiver. Forexample, in a subject experiencing the onset of a seizure or insituations where a seizure has commenced, the subject may requireintrapulmonary administration of the benzodiazepine by a caregiver. Ifthe benzodiazepine is administered in an efficacious amount, theseizure, along with the subject's symptoms of the seizure, shouldrapidly terminate or abort if the seizure does not terminate, a seconddose of the benzodiazepine can be administered.

7. Kits

The pharmaceutical compositions and neurosteroid and benzodiazepinecombinations can be provided in a kit. In certain embodiments, a kit ofthe present invention comprises one or more benzodiazepines and one ormore neurosteroids in separate formulations. In varying embodiments, oneor both of the benzodiazepine and the neurosteroid are provided insubtherapeutic doses or amounts in certain embodiments, the kitscomprise one or more benzodiazepines and one or more neurosteroidswithin the same formulation. In varying embodiments, one or both of thebenzodiazepine and the neurosteroid are provided in subtherapeutic dosesor amounts. In certain embodiments, the kits provide the one or morebenzodiazepines and one or more neurosteroids independently in uniformdosage formulations throughout the course of treatment. In varyingembodiments, one or both of the benzodiazepine and the neurosteroid areprovided in subtherapeutic doses or amounts. In certain embodiments, thekits provide the one or more benzodiazepines and one or moreneurosteroids in graduated dosages over the course of treatment, eitherincreasing or decreasing, but usually increasing to an efficaciousdosage level, according to the requirements of an individual. In varyingembodiments, one or both of the benzodiazepine and the neurosteroid areprovided in subtherapeutic doses or amounts.

In some embodiments, the benzodiazepine is selected from the groupconsisting of bretazenil, clonazepam, cloxazolam, clorazepate, diazepam,fludiazepam, flutoprazepam, lorazepam, midazolam, nimetazepam,nitrazepam, phenazepam, temazepam and clobazam. In some embodiments, thebenzodiazepine is selected from the group consisting of midazolam,lorazepam and diazepam. In some embodiments, the neurosteroid isselected from the group consisting of allopregnanolone,allotetrahydrodeoxycorticosterone, ganaxolone, alphaxolone, alphadolone,hydroxydione, minaxolone, and Althesin. In some embodiments, theneurosteroid is allopregnanolone. In some embodiments, the kit comprisesallopregnanolone and a benzodiazepine selected from the group consistingof midazolam, lorazepam, and diazepam.

In some embodiments, the kits further comprise a NMDA receptorantagonist. In some embodiments, the NMDA receptor antagonist isselected from the group consisting of dizocilpine (MK-801), meperidine,methadone, dextropropoxyphene, tramadol, ketobemidone, ketamine,dextromethorphan, phencyclidine, nitrous oxide (N₂O), AP5(R-2-amino-5-phosphonopentanoate), AP7 (2-amino-7-phosphonoheptanoicacid), CPPene (3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonicacid), selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,eticyclidine, gacyclidine, ibogaine, magnesium, memantine,methoxetamine, rolicyclidine.tenocyclidine, methoxydine, tiletamine,xenon, neramexane, eliprodil, etoxadrol, dexoxadrol, WMS 2539. NEFA,remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211, rhynchophylline,1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA(5,7-dichlorokynurenic acid), kynurenic acid, lacosamide, CP-101,606(traxoprodil), AZD6765 (lanicemine) and GLYX-13. In some embodiments,the NMDA receptor antagonist is selected from the group consisting ofketamine, dextromethorphan, phencyclidine, CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid),selfotel, amantadine, dextrorphan, memantine, tiletamine, neramexane,eliprodil, remacemide, aptiganel, 1-Aminocyclopropanecarboxylic acid(ACPC), 7-Chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenicacid, CP-101,606 (traxoprodil), AZD6765 (lanicemine) and GLYX-13. Insome embodiments, the NMDA receptor antagonist is dizocilpine (MK-801).

In some embodiments, one or both of the benzodiazepine and theneurosteroid is formulated for inhalational, intranasal orintrapulmonary administration. In some embodiments, one or both of thebenzodiazepine and the neurosteroid is formulated for oral or parenteraldelivery. In some embodiments, one or both of the benzodiazepine and theneurosteroid are formulated for a parenteral route selected from thegroup consisting of inhalational, intrapulmonary, intranasal,intramuscular, subcutaneous, transmucosal and intravenous. In someembodiments, the benzodiazepine is an agonist of the benzodiazepinerecognition site on GABA_(A) receptors and stimulates endogenousneurosteroid synthesis. In some embodiments, the neurosteroid issuspended or dissolved in a cyclodextrin (e.g., an α-cyclodextrin, aβ-cyclodextrin or a γ-cyclodextrin). In varying embodiments, theneurosteroid is suspended or dissolved in a cyclodextrin selected fromthe group consisting of hydroxypropyl-β-cyclodextrin, endotoxincontrolled β-cyclodextrin sulfobutyl ethers, or cyclodextrin sodiumsalts (e.g., CAPTISOL®). In some embodiments, the neurosteroid issuspended or dissolved in an edible oil. In some embodiments, the edibleoil comprises one or more vegetable oils. In some embodiments, thevegetable oil is selected from the group consisting of coconut oil, cornoil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil,canola oil, safflower oil, sesame oil, soybean oil, sunflower oil, andmixtures thereof. In some embodiments, the edible oil is canola oil. Insome embodiments, the edible oil comprises one or more nut oils. In someembodiments, the nut oil is selected from the group consisting of almondoil, cashew oil, hazelnut oil, macadamia oil, mongongo nut oil, pecanoil, pine nut oil, pistachio oil, walnut oil, and mixtures thereof.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Tetramethylenedisulfotetramine Alters Ca2+ Dynamics inCultured Hippocampal Neurons: Mitigation by NMDA Blockade and GABA_(A)Receptor Positive Modulation

Materials and Methods

Materials

Fetal bovine serum and soybean trypsin inhibitor were obtained fromAtlanta Biologicals (Norcross, Ga.). DNase, poly-L-lysine, cytosinearabinoside,(−)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate(MK-801), Hydroxypropyl-β-cyclodextran, and (3,5-dimethyl2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate(nifedipine) were from Sigma-Aldrich (St. Louis, Mo.). The Ca2|fluorescence dye Fluo-4, Pluronic F-127 and Neurobasal medium werepurchased from Life Technology (Grand Island, N.Y.).Tetramethylenedisulfotetramine (TETS) was synthesized as describedpreviously (Zolkowska et al., 2012). Diazepam was from Western MedicalSupply (Arcadia, Ca.). Allopregnanolone(3α-hydroxy-5α-pregnan-20-one; >99%) was provided by M A. Rogawski.

Primary Cultures of Hippocampal Neurons.

Animals were treated humanely and with regard for alleviation ofsuffering according to protocols approved by the Institutional AnimalCare and Use Committee of the University of California, Davis.Hippocampal neuron cultures were dissociated from hippocampi dissectedfrom C57BL/6J mouse pups at postnatal day 0-1 and maintained inNeurobasal complete medium [Neurobasal medium supplemented with NS21,0.5 mM L-glutamine, HEPES] with 5% fetal bovine serum. For Ca2| imagingstudies using FLIPR, dissociated hippocampal cells were plated ontopoly-L-lysine coated clear-bottom, black wall, 96-well imaging plate(BD, Franklin Lakes, N.J., USA) at a density of 0.8×10⁵/well. Formicroelectrode array (MEA) experiments, 120 μl of cell suspension at adensity of 1.5×10⁶ cells/ml were added to a 12-well Maestro plate (AxionBiosystems, Atlanta, Ga.). After 2 h incubation, a volume of 1.0 ml ofserum-free Neurobasal complete medium was added to each well. The mediumwas changed twice a week by replacing half volume of culture medium withscrum-free Neurobasal complete medium. The neurons were maintained at37° C. with 5% CO2 and 95% humidity.

Measurement of Synchronous Intracellular Ca2 Oscillations.

Hippocampal neurons between 13-17 days in vitro (DIV) were used toinvestigate how TETS alters synchronous Ca2· oscillations that normallyoccur in healthy neurons at this developmental stage. This methodpermits simultaneous measurements of intracellular Ca2· transients in a96-well format as described as previously (Cao et al., 2010). Baselinerecording were acquired in Locke's buffer (8.6 mM HEPES, 5.6 mM KCl, 154mM NaCl, 5.6 mM glucose, 1.0 mM MgCl₂, 2.3 mM CaCl₂, and 0.0001 mMglycine, pH 7.4) for 10 min followed by addition of TETS and/orpharmacological agents using a programmable 96-channel pipetting roboticsystem, and the intracellular Ca2− was monitored for an additional 30min. Unless otherwise indicated, pharmacological interventions wereintroduced 10 min prior to TETS. TETS triggered an immediate rise in[Ca2·]i that was analyzed by quantifying the Area Under the Curve (AUC;in arbitrary fluorescence units) of the Fluo-4 fluorescence units for aduration of 5 min following TETS addition. TETS also altered thefrequency and amplitude of neuronal synchronous Ca2| oscillations, whichwere analyzed during the 10 min period after addition of TETS.

MEA Recording.

All MEA recordings were conducted at 37° C. in culture medium withoutperfusion using a 12-well Maestro system (Axion BioSystems, Atlanta,Ga.). Each well contains 64 electrodes (30 μm diameter) in an 8×8 gridwith inter-electrode spacing of 200 μm. Before recording basalelectrical activity, the cultures were equilibrated in freshly prepared,pre-warmed neurobasal complete medium for 1 h. The 12-well Maestroplates were loaded onto a temperature regulated headstage containing therecording amplifier and raw extracellular electrical signals wereacquired using Axis software (Axion BioSystems, Atlanta, Ga.). Signalsfrom the amplifier were digitized at a rate of 25 KHZ, and filteredusing Butterworth Band-pass filter (cutoff frequency of 300 Hz). TheAxis software was used to detect spontaneous events that exceeded athreshold of six times of the noise. Raster plot and spike rate analysisdata were performed by exporting the raw data to the NeuroExplorersoftware (version 4.0, NEX Technologies, Littleton, Mass.).

Data Analysis. Graphing and statistical analysis were performed usingGraphPad Prism software (Version 5.0, GraphPad Software Inc., San Diego,Calif.). EC50 values were determined by non-linear regression using athree-parameter logistic equation. Statistical significance betweendifferent groups was calculated using Student's 1-test or by an ANOVAand, where appropriate, a Dunnett's Multiple Comparison Test; p valuesbelow 0.05 were considered statistically significant.

Results

Effects of TETS on Ca2 · Oscillations in Primary Cultured HippocampalNeurons.

Cultured hippocampal neurons (13-17 DIV) exhibit spontaneous synchronousCa2− oscillations whose frequency and amplitude can be quantitativelyassessed in real time using FLIPR® (FIG. 1A). Addition of DMSO vehiclehad no significant effect on the properties of the synchronous Ca2·oscillations during the 5 min Phase I period or the 10 min Phase IIperiod (FIG. 1A, top trace). By contrast, exposure of the neurons toTETS caused an immediate increase in the amplitude of the oscillationsand at higher concentrations (3 and 10 μM) a sustained plateau responsethat decayed slowly over the 5 min Phase I period. The integrated Ca2−signal (area under the curve: AUC) during the Phase I period exhibited aconcentration-dependent increase, with an EC50 value of 2.7 μM [95%confidence interval (95% CI): 1.4-5.2 μM] (FIG. 1B). During Phase II,TETS caused a concentration-dependent decrease in the frequency of thesynchronous Ca2+ oscillations with an EC50 value of 1.7 μM (95% CI0.69-4.12 μM; FIG. 1C) Along with the reduction in frequency, TETSincreased the mean Ca2· oscillation amplitude with an EC50 value of 1.8μM (95% CI: 1.12-2.80 μM; FIG. 1D). TETS modestly prolonged the meanduration of individual Ca2· transients compared to that measured fromvehicle-exposed control neurons. TETS-induced phase II Ca2| responses(both frequency and amplitude) were reversible (FIG. 2)

For comparison, we studied the influence of picrotoxin (PTX; 100 μM), anoncompetitive blocker of GABA_(A) receptor and bicuculline (100 μM), acompetitive antagonist of GABA_(A) receptors on the Ca2| dynamics. Bothantagonists elicited similar Phase I and Phase II responses as TETS(FIG. 3).

TETS Enhances Neuronal Electric Network Activity in Primary CulturedHippocampal Neurons.

Extracellular recordings of electrical activity from multiple siteswithin the neuronal cultures at a high spatial resolution provide arobust measure of network activity and connectivity (Johnstone et al.,2010) After recording the basal electrical activity for 10 min,increasing concentrations of TETS were serially introduced into thewells. The recording was continued for 10 min at each TETS concentrationA control well was simultaneously recorded following introduction ofDMSO vehicle (0.01-0.1%). Basal recordings for up to 60 min showed thatnetwork firing activity was stable in the absence or presence of DMSOvehicle control (FIG. 4A, left panel). Exposure to TETS concentrationsof 2 μM and greater produced a dramatic change in discharge pattern.Events became more highly clustered (FIG. 4A, right panel and FIG. 5)and the duration of clustered bursts induced by 6 μM TETS can last up to10 s (FIG. 4A, right panel, 4th row). There was an overall increase inthe discharge rate (FIG. 4B.) After washout of TETS, the neuronalnetwork firing recovered to basal conditions.

NMDA Receptors, but not L-type Ca2· Channels Are Required forTETS—Induced Ca2· Dysregulation.

We next examined the possible involvement of NMDA receptors and L-typeCa2+ channels in the effects of TETS on Ca2− dynamics. Preincubation ofneuronal cultures for 10 min with MK-801 (1 μM), an NMDA receptorblocker, attenuated both Phase I and Phase II effects of TETS (FIG.6B-D) MK-801 slightly suppressed basal Ca2· oscillations, which isconsistent with an earlier report (Tanaka et al., 1996). By contrast,nifedipine (1 μM), which inhibits L-type voltage activated Ca2|channels, was without effect on TETS-induced Phase I or Phase II Ca2|responses (FIG. 6B-D). These results indicate that NMDA receptors butnot L-type Ca2· channels are required for the effects of TETS on Ca2|fluctuations.

Diazepam and Allopregnanolone Partially Mitigate TETS-Induced Ca2·Dysregulation.

We next determined if the GABA_(A) receptor positive modulators diazepamand allopregnanolone could protect against TETS-induced Ca2|dysregulation. FIG. 7A (top trace) demonstrates that the oscillatoryactivity of neurons exposed to vehicle remained stable over the entirerecording period. Introduction of diazepam (0.1, 0.3, or 1 μM) caused anattenuation in the amplitude of basal spontaneous Ca2+ oscillations(FIG. 7A) Pre-exposure to diazepam caused a smallconcentration-dependent reduction of the Phase I integrated rise in Ca2+induced by TETS that reached statistical significance only at 1 μM (FIG.7B). Diazepam did not eliminate the Phase I plateau response (FIG. 7A).Diazepam also caused a partial inhibition of the Phase II frequency andamplitude effects of TETS, with the effect on amplitude reachingsignificance at 0.1 μM (FIG. 7C, D).

As shown in FIG. 8, allopregnanolone similarly attenuated the effects ofTETS on Ca2| dysregulation. Allopregnanolone (0.1-1 μM) caused aconcentration-dependent suppression of basal spontaneous Ca2·fluctuations and it partially attenuated the response in Phase I at 1 μMwithout eliminating the plateau in Ca2| levels (FIG. 8A, B).Allopregnanolone at 0.3 and 1 μM also inhibited the Phase II effect ofTETS on frequency and amplitude with a completely reversed Phase IIeffect on amplitude at 1 μM (FIG. 8C,D).

Low Concentrations of Diazepam and Allopregnanolone in CombinationMitigate TETS-Induced Ca2· Dysregulation.

We next evaluated the effect of a combination of diazepam andallopregnanolone, each at a low concentration (0.1 μM) that by itselfhas minimal effects on Phase I or Phase II Ca2· dysregulation. As shownin FIG. 9, the combination they strongly mitigated both Phase I andPhase II effects. In fact, the combination treatment was able to largelyeliminate the plateau response obtained with acute TETS exposure (FIG.9A), an effect not obtained with 10-fold higher concentrations ofdiazepam (FIG. 7) or allopregnanolone (FIG. 8) alone.

Discussion

In the present study, we characterized the effects of TETS onhippocampal neurons in culture using MEA field potential recording andFluo-4 fluorescence measurements of Ca2| dynamics in the neuronalnetwork. Over time, hippocampal neurons in culture develop a richnetwork of processes and form numerous functional synaptic contacts(Mennerick et al., 1995; Arnold et al., 2005). Cultures that havedeveloped for 13-17 DIV as used in the present study are well organizedand there is robust spontaneous electrical activity mediated byexcitatory and inhibitory transmission between neurons. Neurons withinthe cultures exhibit spontaneous action potentials and cultures ofsufficient cell density may show synchronized bursting of neuronsthroughout the entire culture (Arnold et al., 2005). Excitatory synaptictransmission is mediated by functional glutamate receptors of the NMDAand AMPA types (Abele et al., 1990). Importantly, the cultures containGABAergic neurons, comprising approximately 10 percent of the neurons,that form robust inhibitory synaptic connections mediated by GABA_(A)receptors (Jensen et al., 1999; Jensen et al., 2000). Inhibitorysynaptic potentials in hippocampal cultures have physiologicalproperties that are similar to those obtained in intact preparations(Jensen et al., 1999) The GABAergic neurons impose tonic inhibition ontothe network so that exposure of hippocampal cultures to GABA_(A)receptor antagonists causes increased action potential firing,spontaneous rhythmic neuronal depolarizations, and bursting. Therhythmic depolarizations and bursting is dependent upon actionpotentials as it is eliminated by tetrodotoxin.

MEA recording allow the electrical activity of multiple neurons withinthe cultures to be monitored whereas FLIPR® Fluo-4 fluorescencemeasurements provide a dynamic assessment of aggregate intracellularCa2| levels (Cao et al., 2010; Cao et al., 2012). Using these assays, wefound that TETS dramatically increases intracellular Ca2· levels andalters Ca2| dynamics, initially causing an transient increase on theintracellular Ca2· concentration ([Ca2·]i) followed by a decrease on theCa2· oscillations having bigger amplitude. Assessment of ongoingelectric activity in the cultures with MEA recording showed an overallincrease in discharge frequency and a change in the pattern of thedischarges to clustering followed by periods of electrical silence. Theactions of TETS on neuronal Ca2− dynamics and electrical dischargeactivity occur within the same concentration range, suggesting the twoeffects are mechanistically linked. TETS-induced changes on Ca2·dynamics and on electrical discharges are similar to those observed withthe GABA_(A) receptor antagonists bicuculline or picrotoxin (Arnold etal., 2005; Cao et al., 2012). Additionally, TETS modulation of Ca2·dynamics and spontaneous neuronal firing activity in aconcentration-dependent manner with EC50 values of approximately 1-2 μMwhich is consistent with the affinity of TETS for GABA_(A) receptors(Bowery et al., 1975; Dray, 1975, Roberts et al., 1981). Collectivelythese data support the view that the GABA_(A) receptor blocking activityof TETS is responsible for the effects. Like picrotoxin, TETS isbelieved to be a reversible inhibitor of GABA_(A) receptors, which isalso consistent with the rapid reversibility of its effects in the MEAassay.

TETS-triggered alterations in electric firing and synchronous Ca2+oscillations appear to rely on spontaneous action potentials since theyare prevented by tetrodotoxin block of Na+ channels. The neuronalspecificity of TETS in producing both Phase I and Phase II Ca2·responses in hippocampal cultures is also indicated by the observationsthat addition of TETS up to 3 μM to the culture medium of skeletalmyotubes alters neither basal Ca2· homeostasis nor electrically evokedCa2· transients (i.e., excitation-contraction coupling).

A key observation in the present study is that the alterations in Ca2|dynamics induced by TETS was largely inhibited by MK-801 demonstratingthat NMDA receptors are required. While direct activation of NMDAreceptors by TETS is not excluded, activation of NMDA receptors by bathapplication of NMDA increase the neuronal firing in a evenly distributedpattern which is not similar to the clustered bursts firng elicited byTETS or other GABA_(A) receptors blocker/antagonist such as picrotoxin(Cao et al., 2012) The NMDA receptor dependence for TETS response to theCa2− is consistent with earlier evidences in vivo that the NMDA receptorantagonist MK-801 inhibits picrotoxin or bicuculline-induced convulsionin mice (Obara, 1995; Czlonkowska et al., 2000) and ex vivo that theNMDA antagonist 2-APV suppresses picrotoxin-induced Ca2· responses aswell as the frequency and duration of the epileptiform discharges inhippocampal slice preparation (Kohr and Heinemann, 1989). How thesuppression of GABA_(A) receptors activity by TETS affects NMDA receptorfunctions remains to be established. One possibility is that the Phase I[Ca2+]i response may involve presynaptic glutamate transmission. Insupport, bicuculline-induced [Ca2−]i responses have been shown toinvolve synaptic but not-extra-synaptic NMDA receptor activation(Hardingham et al., 2001; Hardingham et al., 2002). While therelationship between the Ca2| signals in the rapid throughput FLIPRassay and epileptic activity remain to be determined, our observationthat NMDA receptors are required for the TETS-induced changes in Ca2·dynamics supports the concept that the effects on Ca2| are a surrogatefor epileptic activity and may be useful as a model for therapeuticsdiscovery. This is further supported by our demonstration that GABA_(A)receptors positive allosterical enhancer, diazepam or allopregnanolonepartially suppress TETS-induced modulation of Ca2+ dynamics.

Consistent with the role of GABA_(A) receptors in restraining burstingand altered Ca2| dynamics is our observation that the GABA_(A) receptorpositive modulators diazepam and allopregnanolone are able to protectagainst the effects of TETS on Ca2+ dynamics. Allopregnanolone was moreeffective on mitigation of Phase I response induced by TETS thandiazepam. This is consistent with the fact that diazepam only acts onsynaptic GABA_(A) receptors, whereas neurosteroids such asallopregnanolone can enhance both extrasynaptic synaptic GABA_(A)receptors (Kokate et al., 1994; Lambert et al., 2003; Reddy andRogawski, 2012). However, neither diazepam nor allopregnanolone alonewas fully effective, even at the highest concentrations tested (1 μM).Unexpectedly, we found that the combination of diazepam andallopregnanolone, each at a threshold concentration of 0.1 μM, washighly effective at protecting against the effects of TETS on Ca2+dynamics, causing a nearly complete inhibition of the Phase I response,including the plateau in Ca2−, as well as the Phase II changes. Thecombination of a benzodiazepine and a neurosteroid has not to ourknowledge previously been studied in a simplified functional system. Itis well recognized that benzodiazepines such as diazepam only act onsynaptic GABA_(A) receptors, whereas neurosteroids such asallopregnanolone preferentially enhance extrasynaptic GABA_(A) receptorsalthough they also act on synaptic receptors as well (Kokate et al.,1994; Lambert et al., 2003; Reddy and Rogawski, 2012). Without beingbound to theory, it appears that the combined action on synaptic andextrasynaptic receptors accounts for the unique potency of the drugcombination.

Alternatively, there may be an interaction at the level of individualGABA_(A) receptors. The recognition sites for neurosteroids on GABA_(A)receptors are distinct from those that recognize benzodiazepines andbarbiturates (Johnston, 1996). It is conceivable, however, thatallopregnanolone and diazepam could produce a synergistic enhancement ofGABA_(A) receptors in a similar fashion as the synergism that occursbetween barbiturates and benzodiazepines, where there is known to beallosteric coupling (DeLorey et al., 1993).

In summary, we have developed rapid throughput methods to detectTETS-induced Ca2| dysregulation and altered electrical activity incultured hippocampal neurons. We demonstrated that two GABA_(A)receptors allosteric modulators, allopregnanolone and diazepam, whenintroduced singly prior to TETS, mitigate TETS-induced Ca2·dysregulation, demonstrating that the in vitro methods described herehave translational value to identify new therapies and optimizecombinatorial strategies for the prevention of TETS poisoning. The basicapproaches described here are of general utility for investigatingchemically diverse threat agents that elicit changes in the electricalbehavior or Ca2− dynamics of in vitro neuronal networks. These rapidthroughput approaches are useful for identifying novel targetedinterventions and for optimizing therapeutic strategies involving drugcombinations.

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Example 2 Combination Treatment with a Benzodiazepine and a NeurosteroidMitigates the Severity and Prevents the Lethality of Seizures Even whenAdministering after Seizure has Started

In cultured hippocampal neurons, higher concentration of TETS (>3 μM)produces an acute elevation of intracellular Ca2| levels (Phase Iresponse) and a prolonged Ca2· response with increased Ca2· oscillationamplitude and decreased frequency of the Ca2· oscillations (Phase IIresponse). Both Phase I and Phase II response can be mitigated by thepretreatment of diazepam and allopregnanolone. More importantly,pre-treatment with a combination of low concentrations of diazepam andallopregnanolone, which have minimal effect against TETS-induced Ca2+response, normalized the TETS Ca2+ response to the control level (Cao etal., Toxicological Sciences, 130: 362-372). In this study, we examinedwhether post-treatment (after TETS triggers Phase I and Phase II Ca2·responses) of neurons with diazepam and/or allopregnanolone mitigatealterations triggered by TETS, which is more relevant to the TETSpoising. Since addition of TETS induces acute phase I response, wetherefore only focused on the TETS-induced Phase II response. Additionof vehicle (0.1% DMSO) was no effect on the Ca2| dynamics over therecording period of 45 min. However, a concentration of 3 μM of TETSproduced an acute Phase I and a prolonged Phase II effect, as previouslyreported (Cao et al 2012) While addition of diazepam (0.1 μM) orallopregnanolone (0.1 μM) singly was without significant effect onTETS-induced decreased Ca2+ oscillation frequency, diazepam (0.1 μM) andallopregnanolone (0.1 μM) in combination effectively recoveredsynchronous Ca2| oscillations characteristics comparable to thoseobserved with vehicle-treated cultures. Although allopregnanolone (0.1μM) alone decreased the TETS-induced Ca2· oscillation amplitude ˜20%(p<0.01), the post-TETS treatment with diazepam (0.1 μM) andallopregnanolone (0.1 μM) in combination conferred much greater recoveryof Ca2-oscillation amplitude to that below vehicle control, and occurredrapidly after the addition of diazepam and allopregnanolone (FIG. 10).These data clearly demonstrate that diazepam in combination with aneurosteroid, such as allopregnanolone, act in a synergistic manner tomitigate the severity of seizures and prevent the lethality ofseizurogenic agents After the seizures have already started.

Example 3 In Vivo Assay Demonstrating the Therapeutic Efficacy ofCombined Benzodiazepine and Neurosteroid in Mitigating TETS-InducedSeizures and Death

When administered to adult male NIII Swiss mice at lethal doses, TETStypically causes two clonic seizures within the first 20 minutes afterTETS injection with each seizure lasting approximately 30 to 45 seconds.These clonic seizures are followed by a tonic seizure that results inthe death of >95% of the TETS-intoxicated animals (FIG. 11). Mice can berescued from TETS-induced death if they are administered a very highdose of diazepam (5 mg/kg, i.p.) immediately following the second clonicseizure (FIG. 11). Administration of diazepam at 0.03 mg/kg immediatelyfollowing the second clonic seizure protected <10% of theTETS-intoxicated animals from death (FIG. 12). Pretreatment withdiazepam (0.1 mg/kg 10 minutes before TETS injection) protected <30% ofthe TETS-intoxicated animals from death (FIG. 13) Post-administration ofthe neurosteroid allopregnanolone at 0.03 mg/kg was no more efficaciousthan low dose diazepam in protecting TETS-intoxicated animals from death(FIG. 12). Pretreatment with allopregnanolone at 0.1 mg/kg protected˜50% of the TETS-intoxicated animals (FIG. 13). When administeredsimultaneously, the subthreshold doses of diazepam and allopregnanolonesignificantly increased survival of TETS-intoxicated animals. Whenadministered immediately after the second clonic seizure, thisbenzodiazepine and neurosteroid combination, 100% of theTETS-intoxicated animals survived (FIG. 12). Used as a pretreatment,this combinatorial therapy protected ˜75% of the TETS-intoxicatedanimals (FIG. 13). Importantly, the therapeutic combination ofsubthreshold diazepam and allopregnanolone had no effect on bloodpressure, whereas the dose of diazepam required to prevent TETS-inducedtonic seizures and death when administered singly caused significanthypotension (FIG. 14).

The significance of these in vivo data are 3-fold: (1) these dataconfirm the predictive value of the in vitro screening system; (2) thesedata demonstrate that combinatorial therapy with a benzodiazepine and aneurosteroid, at subthreshold doses that singly have no effect, isefficacious in preventing seizures and in the case of TETS, inpreventing death associated with tonic seizures; and (3) thecombinatorial therapy avoids an important off-target adverse effect(significant decrease in blood pressure) associated with use of diazepamwhen used singly at therapeutic doses.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of preventing or mitigating a seizure ina subject in need thereof, comprising: (a) determining that a subject isat risk of being exposed to a nerve agent or pesticide that can causeseizures, and (b) administering to the subject of an effective amount ofa benzodiazepine and allopregnanolone, wherein each of thebenzodiazepine and the allopregnanolone are administered in asubtherapeutic dose, and wherein the subtherapeutic dose of thebenzodiazepine is in the range of 0.3 μg/kg to 3.0 μg/kg.
 2. The methodof claim 1, wherein the agent or pesticide is an organophosphorus nerveagent or pesticide.
 3. The method of claim 1, wherein the nerve agent orpesticide is selected from the group consisting of tabun, sarin, soman,GF, VR, VX, Acephate (Orthene), Azinphos-methyl (Gusathion, Guthion),Bensulide (Betasan, Lescosan), Bomyl (Swat), Bromophos (Nexion),Bromophos-ethyl (Nexagan), Cadusafos (Apache, Ebufos, Rugby),Carbophenothion (Trithion), Chlorethoxyfos (Fortress), Chlorfenvinphos(Apachlor, Birlane), Chlormephos (Dotan), Chlorphoxim (Baythion-C),Chlorpyrifos (Brodan, Dursban, Lorsban), Chlorthiophos (Celathion),Coumaphos (Asuntol, Co-Ral), Crotoxyphos (Ciodrin, Cypona), Crufomate(Ruelene), Cyanofenphos (Surecide), Cyanophos (Cyanox), Cythioate(Cyflee, Proban), DEF (De-Green), Demeton (Systox), Demeton-S-methyl(Duratox, Metasystoxl), Dialifor (Torak), Diazinon, Dichlorofenthion,(VC-13 Nemacide), Dichlorvos (DDVP, Vapona), Dicrotophos (Bidrin),Dimefos (Hanane, Pestox XIV), Dimethoate (Cygon, DeFend), Dioxathion(Delnav), Disulfoton (Disyston), Ditalimfos, Edifenphos, Endothion, EPBP(S-seven), EPN, Ethion (Ethanox), Ethoprop (Mocap), Ethyl parathion(E605, Parathion, thiophos), Etrimfos (Ekamet), Famphur (Bash, Bo-Ana,Famfos), Fenamiphos (Nemacur), Fenitrothion (Accothion, Agrothion,Sumithion), Fenophosphon (Agritox, trichloronate), Fensulfothion(Dasanit), Fenthion (Baytex, Entex, Tiguvon), Fonofos (Dyfonate,N-2790), Formothion (Anthio), Fosthietan (Nem-A-Tak), Heptenophos(Hostaquick), Hiometon (Ekatin), Hosalone (Zolone), IBP (Kitazin),Iodofenphos (Nuvanol-N), Isazofos (Brace, Miral, Triumph), Isofenphos(Amaze, Oftanol), Isoxathion (E-48, Karphos), Leptophos (Phosvel),Malathion (Cythion), Mephosfolan (Cytrolane), Merphos (Easy Off-D,Folex), Methamidophos (Monitor), Methidathion (Supracide, Ultracide),Methyl parathion (E601, Penncap-M), Methyl trithion, Mevinphos(Duraphos, Phosdrin), Mipafox (Isopestox, Pestox XV), Monocrotophos(Azodrin), Naled (Dibrome), Oxydemeton-methyl (Metasystox-R),Oxydeprofos (Metasystox-S), Phencapton (G 28029), Phenthoate(Dimephenthoate, Phenthoate), Phorate (Rampart, Thimet), Phosalone(Azofene, Zolone), Phosfolan (Cylan, Cyolane), Phosmet (Imidan,Prolate), Phosphamidon (Dimecron), Phostebupirim (Aztec), Phoxim(Baythion), Pirimiphos-ethyl (Primicid), Pirimiphos-methyl (Actellic),Profenofos (Curacron), Propetamphos (Safrotin), Propyl thiopyrophosphate(Aspon), Prothoate (Fac), Pyrazophos (Afugan, Curamil), Pyridaphenthion(Ofunack), Quinalphos (Bayrusil), Ronnel (Fenchlorphos, Korlan),Schradan (OMPA), Sulfotep (Bladafum, Dithione, Thiotepp), Sulprofos(Bolstar, Helothion), Temephos (Abate, Abathion), Terbufos (Contraven,Counter), Tetrachlorvinphos (Gardona, Rabon), Tetraethyl pyrophosphate(TEPP), tetramethylenedisulfotetramine (TETS), Triazophos (Hostathion),and Trichlorfon (Dipterex, Dylox, Neguvon, Proxol).
 4. The method ofclaim 1, wherein the nerve agent or pesticide istetramethylenedisulfotetramine (TETS).
 5. The method of claim 1, whereinthe benzodiazepine and the allopregnanolone are co-administered togetherand/or by the same route of administration.
 6. The method claim 1,wherein the benzodiazepine and the allopregnanolone are co-administeredseparately and/or by different routes of administration.
 7. The methodclaim 1, wherein one or both of the benzodiazepine and theallopregnanolone are administered by the inhalational or intrapulmonaryroute of administration.
 8. The method of claim 7, wherein thebenzodiazepine and the allopregnanolone are not heated prior toadministration.
 9. The method of claim 7 or 8, wherein one or both ofthe benzodiazepine and allopregnanolone are aerosolized.
 10. The methodclaim 9, wherein one or both of the benzodiazepine and theallopregnanolone aerosolized particulates having a mass medianaerodynamic diameter ranging from about 1μm to about 3μm.
 11. The methodof claim 1, wherein the allopregnanolone is administered at a dose inthe range of about 5 mg/kg to about 50 mg/kg.
 12. The method of claim 1,wherein the benzodiazepine is selected from the group consisting ofbretazenil, clonazepam, cloxazolam, clorazepate, diazepam, fludiazepam,flutoprazepam, lorazepam, midazolam, nimetazepam, nitrazepam,phenazepam, temazepam and clobazam.
 13. The method of claim 1, furthercomprising the co-administration of an NMDA receptor antagonist.
 14. Themethod of claim 13, wherein the NMDA receptor antagonist is selectedfrom the group consisting of dizocilpine (MK-801), meperidine,methadone, dextropropoxyphene, tramadol, ketobemidone, ketamine,dextromethorphan, phencyclidine, nitrous oxide (N₂O), APS(R-2-amino-5-phosphonopentanoate), AP7 (2-amino-7-phosphonoheptanoicacid), CPPene (3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonicacid), selfotel, amantadine, dextrallorphan, dextrorphan, ethanol,eticyclidine, gacyclidine, ibogaine, magnesium, memantine,methoxetamine, rolicyclidine.tenocyclidine, methoxydine, tiletamine,xenon, neramexane, eliprodil, etoxadrol, dexoxadrol, WMS 2539, NEFA,remacemide, delucemine, 8A-PDHQ, aptiganel, HU-211, rhynchophylline,1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate, DCKA(5,7-dichlorokynurenic acid), kynurenic acid, lacosamide, CP-101,606(traxoprodil), AZD6765 (lanicemine) and GLYX-13.
 15. The method of claim13, wherein the benzodiazepine, allopregnanolone and NMDA receptorantagonist are co-administered together and/or by the same route ofadministration.
 16. The method of claim 13, wherein the benzodiazepine,allopregnanolone and NMDA receptor antagonist are administeredseparately and/or by different routes of administration.